Tertiary amines for use in the treatment of cardiac disorders

ABSTRACT

The present invention relates to tertiary amines of formula (I) for use in therapy, particularly for use in treating cardiovascular disorders. The compounds have been found to regulate phospholamban phosphorylation by interfering with the A-kinase anchor protein 18delta (AKAP18δ) binding to the PKA substrate phospholamban. The compounds share a tri(alkylaryl/alkylheteroaryl) amine structure.

The invention relates to new chemical compounds, new pharmaceuticalcompositions and use of these compounds and their compositions fortreatment of cardiac disorders especially reperfusion syndrome followingmyocardial infarction and post-infarction chronic heart failure. Thespecific compounds interfere with the A-kinase anchor protein (AKAP)18delta (AKAP18δ) binding to the PKA substrate phospholamban.

The A-kinase anchor proteins (AKAPs) are a group of structurally diverseproteins which have the common function of binding to the regulatorysubunit of protein kinase A (PKA) and confining the PKA holoenzyme todiscrete locations within the cell. There are at least 50 differentAKAPs, all of which have been cloned. Typical AKAPs include for exampleAKAP79, AKAP18, AKAP450, all of which are somewhat arbitrarily namedafter their apparent mobility by SDS-poly acrylamide gelelectrophoresis. Later, the gene nomenclature committee has alsointroduced a separate nomenclature for AKAPs where they areconsecutively numbered AKAP1, AKAP2, AKAP3 etc. In addition, some AKAPslike ezrin and Opa1 have already been assigned other names that are inuse.

AKAPs act as targeting devices that assemble signaling elements on ascaffold (the AKAP) that itself targets to microdomains in cells. Thisallows specific targeting of substrates to be regulated byphosphorylation (by PKA) and dephosphorylation (by phosphatases). PKAbinds via its regulatory subunits (RIα, RIIα, RIβ, RIIβ) directly to anamphipathic α-helix in the AKAP, which is a common feature of AKAPs. TheAKAPs also bind other components including; phosphodiesterases (PDEs)which break down cAMP, phosphatases which dephosphorylate downstream PKAtargets and also other kinases (PKC and MAPK). Some AKAPs are able tobind both regulatory subunits (RI & RII) of PKA and are dual-specificAKAPs (for example D-AKAP1, D-AKAP2, ezrin, OPA1).

For references related the various AKAPs and their biological functionssee for example:

Tasken and Aandahl in Physiological Reviews volume 84, issue 1, page137-167 (2004), Pidoux and Tasken in Journal of Molecular Endocrinologyvolume 44, issue 5, page 271-284 (2010), Feliciello et al in Journal ofMolecular Biology, volume 308, issue 2, page 99-114 (2001), A. McCahillet al. in Cellular Signalling, volume 17, issue 9, page 1158-1173(2005), L. Cardone et al. in Journal of Molecular Biology, volume 320,issue 3, page 663-675, A. M. Sardanelli et al. in FEBS Letters, volume580, issue 24, page 5690-5696 (2006), B. Hu et al. in Biochemical andBiophysical Research Communications, volume 285, issue 5, page 1369-1376(2001), S. Herrgaard et al. in FEBS Letters, volume 486, issue 2, page107-111 (2000), B. Abrenica et al. in Journal of Molecular and CellularCardiology, volume 46, issue 5, 674-681 (2009), O. M. Seternes et al. inCellular Signaling, volume 11, issue 3, page 211-219 (1999), K. Kuriharaet al. in Biochemical Pharmacology, volume 66, issue 2, page 239-250(2003), S. B. Moss et al. in Trends in Endocrinology & Metabolism,volume 12, issue 10, page 434-440 (2001), T. E. Lewis et al. in UrologicOncology: Seminars and Original Investigations, volume 23, issue 6, page407-412 (2005), F. W. Herberg et al, in Journal of Molecular Biology,volume 298, issue 2, page 329-339 (2000), C. R. Carlson et al. inJournal of Molecular Biology, volume 327, issue 3, page 609-618 (2003),R. B. Brown et al. in Biochemical and Biophysical ResearchCommunications, volume 306, issue 2, page 394-401 (2003), T. Kurosu etal. in Brain Research, volume 1251, page 53-64 (2009), M. Hadad et al.in Mechanisms of Development, volume 128, issues 7-10,page 471-482(2011), L. R. Johnson et al. in Development Biology, volume 192, issue2, page 340-350 (1997), D. Diviani et al, in European Journal of CellBiology, volume 85, issue 7, page 603-610 (2006), A. Feliciello et al.in Current Biology, volume 7, issue 12, page 1011-1014 (1997), G. K.Carnegie et al. in Molecular Cell, volume 15, issue 6, 889-899 (2004),A. Tamai et al. in International Congress Series, volume 1283, page263-264 (2005), F. S. Kinderman et al. Molecular Cell, volume 24, issue3, page 397-408 (2006), M. Colledge et al. in Neuron, volume 27,issue 1. page 107-119 (2000), Biochemical and Biophysical ResearchCommunications, volume 225, issue 1, 313-319 ((1996), A. S. Cantrell etal. in Molecular and Cellular Neuroscience, volume 21, issue 1, page63-80, K. Josefsen et al. in FEBS Letters, volume 584, issue 1, page81-85 (2010), P. Klingbell et al. in Mechanisms of Development, volume100. issue 2, page 323-326 (2001), G. K. Carnegie et al. in MolecularCell, volume 32, issue 2, page 169-179 (2008), C. Riether et al. inBrain, Behavior, and Immunity, volume 25, issue 1, page 59-66 (2011), D.Diviani et al, in Current Biology, volume 10, issue 7, page 417-420(2000), J. D. Scott et al in Handbook of Cell Signaling, volume 2, page283-388 (2003), T-T Aye et al. in Journal of Molecular and CellularCardiology, volume 52, issue 2, 511-518 (2012), A. L. Bauman et al. inNeuropharmacology, volume 46, issue 3, page 299-310 (2004), J. D. Scottet al. in Handbook of Cell Signaling (Second Edition), page 1337-1347(2010), M. G. Gold et al. in Molecular Cell, volume 24, issue 3, 383-395(2006), A. Carrera et al. in Development Biology, volume 180, issue 1,page 284-296 (1996), M. L. Del′Acqua et al. in European Journal of CellBiology, volume 85, issue 7, page 627-633 (2006), N. W. Court et al. inBiochimica et Biophysica Acta (BBA)-Molecular Cell Research, volume1744, issue 1, page 68-75 (2005)

There are a so far only a limited number of patent documents describingAKAPs. The most relevant documents are:

US2011/158905 (IBC Pharmaceuticals) describes a fusion proteincomprising an anchoring domain (AD) moiety or a dimerization and dockingdomain (DDD) moiety, wherein the AD moiety consists of the amino acidsequence of the AD moiety of an AKAP (A-kinase anchoring protein) andthe DDD moiety consists of the amino acid sequence of the DDD moiety ofa human protein kinase A regulatory subunit: and an effector moiety. Theeffector moiety is selected from the group consisting of an antibody, anantigen-binding antibody fragment, a cytokine, a viral antigen, axenoantigen, an RNase, a heat shock protein, the N-A1 domain of CEACAM5,the A3-B3 domain of CEACAM5, alpha2-macroglobulin, HSA (human serumalbumin), a human protamine, and Fc fragment of a human antibody and anucleic acid binding protein. The proteins might be useful for treatmentof various diseases or conditions selected from the group consisting ofcancer, autoimmune disease, immune dysregulation disease, organ-graftrejection, graft-versus-host disease, a neurodegenerative disease, ametabolic disease and a cardiovascular disease.

U.S. Pat. No. 7,432,342 (Sequenom) relates to A-kinase anchor protein(AKAPs) muteins, peptides thereof, and nucleic acids encoding thepeptides, especially a polypeptide that is a mutein of a D-AKAF2polypeptide, wherein the mutein exhibits modified binding to aregulatory subunit of PKA compared to a native D-AKAP2.

EP2098226 (Forsungsverbund Berlin E.V.) describes use of bicycliccompounds (I) or their salts, solvates, hydrates or formulations forpreparing a medicament for the prophylaxis or treatment of diseasesassociated with defect of compartmentalized cyclic adenosinemonophosphate (cAMP)-dependent signal transduction, is claimed. Use ofbicyclic compounds of formula (I) or their salts, solvates, hydrates orformulations for preparing a medicament for the prophylaxis or treatmentof diseases associated with defect of compartmentalized cyclic adenosinemonophosphate (cAMP)-dependent signal transduction, is claimed. Formula(X) describes a general bicyclic formula in EP2098226, while formula (I)describes biphenyl compounds which are the most preferred compounds.

A:O, S, NH, CO, (hetero)alkyl, alkenyl, alkynyl, (hetero)aryl,cycloalkylene, (hetero)alkylcycloalkylene, heterocycloalkylene or(hetero)aralkylene group; R, R1:H, halo, NH₂, OH, NO₂, (hetero)alkyl,alkenyl, alkynyl (all preferred), SH, N₃, (hetero)aryl,(hetero)cycloalkyl, (hetero)alkylcycloalkyl or (hetero)aralkyl residue;D, E:(hetero)aryl, (hetero)cycloalkyl, (hetero)alkylcycloalkyl or(hetero)aralkyl (preferably substituted phenyl group); and m, n: 0-5(preferably 1-3). The mechanism of action for these bicyclic compoundsare protein kinease A and as AKAP interaction inhibitor. The indicationsfor these agents are cardiovascular indications like hypertension andvasotropic activity.

WO2007/028969 (University of Oslo) relates to molecules which modify thebinding between AKAP 18d and phosphodiesterase 4D or AKAP 18d andphospholamban and their use in altering PKA type II-mediated, activationof SERCA2 in a cell, for example to alleviate cardiovascular disease.Preferably such molecules include the motif RRASTIE. Molecules such asthose which mimic binding of AKAP 18d to PKA, which allow enhancedphosphorylation of PLB are also discussed. No low-molecular weightcompounds are specifically mentioned in this document.

WO2006/122546 describes non-peptide protein kinase A/protein kinase Aanchor protein decouplers or disruptors. The compounds are listed inextensive tables of compounds. The chemical structures are structurallydiverse and cannot be represented in any general formula. The compoundsdisclosed in these tables do not fall within the general formula ofcompounds of the present invention. The potential indications for use ofthese compounds are very broad including an extensive listing of verymany different diseases, disorders and conditions.

WO2006/154330 (Forsungsverbund Berlin E.V.) relates to a nucleic acidsequence encoding a protein kinase A anchor protein, to the use of saidnucleic acid sequence in a fusion protein, to a method of determiningthe interaction of said protein kinase A anchor protein with regulator/subunits of protein kinase A, and to a method of identifyingcell-permeable substances.

WO2006/032923 (University of Oslo) describes a PKA I anchoringdisrupting molecule or AKAP mimic, wherein said molecule or mimic is apolypeptide which comprises the following amino acid sequence: X1 X2 X3Y A X4 X5 L A X6 X7 X8|X9 X10 X11 X12 X13 (sequence (1)) or apeptidomimetic or analogue thereof is provided. Also provided areantibodies to the molecule, nucleic acid molecules comprising a sequenceencoding the molecule and pharmaceutical compositions. A method ofaltering the PKA type I signaling pathway in a cell by administration ofthe anchoring disruption molecule or AKAP mimic, in particular to treatimmunosuppressive disorders, proliferative diseases or autoimmunediseases is also described.

WO2006/032909 (University of Oslo) describes a PKA II anchoringdisruption molecule or AKAP mimic, wherein said molecule or mimic is apolypeptide which comprises the following amino acid sequence: X1 X2 EX3 X4 AKQ/V X5 X6 X7/X8 X9 X10 (sequence (1)) or a peptidomimetic oranalogue thereof is provided. Also provided are antibodies to themolecule, nucleic acid molecules comprising a sequence encoding themolecule and pharmaceutical compositions. A method of altering the PKAtype II signaling pathway in a cell by administration of the anchoringdisruption molecule or AKAP mimic, in particular to treat cardiovascularand metabolic disorders is also described.

US2009/104177 (Forsungsverbund Berlin E.V.) relates to a nucleic acidsequence encoding peptides which inhibit the interaction of proteinkinase A (PKA) and protein kinase A anchor proteins (AKAP), to a hostorganism comprising said nucleic acid sequence and optionally expressingsaid peptides, to the use of said peptides and of said host organism ininvestigating diseases associated with said AKAP-PKA interaction, and tothe use of said peptides as pharmaceutical agent for the treatment ofsuch diseases.

U.S. Pat. No. 6,958,214 (Sequenom) relates to polymorphic A-kinaseanchor proteins (AKAPs) and nucleic acids encoding the proteins areprovided herein. Methods of detecting polymorphic AKAPs and nucleicacids encoding the AKAPs, and kits for use in the detection methods arealso provided. Further provided herein are methods of identifyingsubjects having or at risk of developing disorders of signaltransduction. Methods of determining susceptibility to morbidity and/orincreased or early mortality are also described.

WO2004/081576 (Sequenom) relates to polymorphic A-kinase anchor proteins(AKAPs) and nucleic acids encoding the proteins are provided herein.Methods of detecting polymorphic AKAPs and nucleic acids encoding theAKAPs, and kits for use in the detection methods are also provided.Further provided herein are methods of identifying subjects having or atrisk of developing diseases or disorders, such as those related tosignal transduction and/or cardiovascular disease. Methods ofdetermining susceptibility to morbidity and/or increased or earlymortality are also described.

U.S. Pat. No. 6,107,104 (ICOS) relates to compositions and methodsuseful for isolating calcineurin as well as inhibiting, calcineurinactivity. The compositions are peptides that contain regions that arehomologous to calcineurin-binding regions of Akap79. Also provided aremethods for determining if a cell contains a calcineurin-binding andPKA-binding anchoring protein that are useful for identifying additionalproteins that bind both calcineurin and PKA. Another aspect of thepresent invention is methods for enhancing expression of interleukin 2by T cells. Further provided are methods to identify proteins whichinteract with AKAP79, and methods to identify inhibitors of AKAP 79interaction with other proteins.

The present invention relates to new chemical compounds, newpharmaceutical compositions and use of compounds and their compositionsfor treatment of cardiac disorders especially cardiac failure. Thespecific compounds interfere with the ability of the A-kinase anchorprotein (AKAP) 18δ to bind to the PKA substrate phospholamban and bydisrupting this interaction specifically inhibits PKA phosphorylation ofphospholamban upon adrenergic stimulation.

One aspect of the present invention relates to specific compoundsinterfering with A-kinase anchor proteins.

Thus, the present invention relates to a compound of formula (I) for usein therapy:

wherein

L¹, L² and L³ independently denote C₁-C₄-alkylene optionally substitutedwith one phenyl;

Ar¹, Ar² and Ar³ independently denote a 5-10 membered heteroaryloptionally substituted with —C(═O)Z or one or more R; or a 6-10 memberedaryl optionally substituted with one or more R;

Z denotes OR^(a) or NR^(b)R^(c);

R^(a) denotes H, C₁-C₄-alkyl, C₃-C₅-cycioalkyl,C₂-C₄-alkylene-O—C₁-C₄-alkyl, C₂-C₄-alkylene-CN; C₁-C₄-alkyl substitutedwith one or two Ar⁴;

R^(b) denotes H, C₁-C₄-alkyl, C₃-C₆-cycloalkyl, C₂-C₄-alkylene-CN;C₂-C₄-alkylene-O—C₁-C₄-alkyl; or C₁-C₄-alkyl substituted with one or twoAr⁴;

R^(c) denotes H, C₁-C₈ alkyl, C₃-C₆-cycloalkyl, Ar⁴, C₁-C₄-alkylsubstituted with one or two Ar⁴; OH, O—-C₁-C₄-alkyl,C₂-C₄-alkyl-O—C₁-C₄-alkyl, or C₂-C₄-alkyl-NR^(a)R^(a);

or together NR^(b)R^(c) denotes

wherein X denotes CHZ¹, O, or NZ¹;

Z¹ denotes H, C(═O)H, C(═O)C₁-C₄-alkyl, C(═O)OR^(z), Ar⁴, C₁-C₄-alkyl,C₃-C₆-cycloalkyl, C₁-C₄-alkyl substituted with one or two Ar⁴;C₂-C₄—NR^(d)R^(e), or C₂-C₄—OR¹;

Z² denotes OR^(a) or NR^(g)R^(g);

Ar⁴ denotes 5-6 membered heteroaryl; or phenyl optionally substitutedwith one or more R;

R^(d), R^(e) and R^(f) independently denote H or C₁-C₄-alkyl;

R^(g) denotes R^(h);

R^(z) denotes H, C₁-C₄-alkyl or C₃-C₆-cycloalkyl;

R independently denotes F, Cl, Br, I, C₁-C₄-haloalkyl, OR¹, SR¹, NO₂,NR²R³, R⁴, C(═O)Y, SO₃H, C₁-C₄-alkyl, C₁-C₄-alkyl-phenyl, a 5-memberedheteroaryl; or a phenyl substituted with a 5-membered heteroaryl;

R¹ denotes H, C₁-C₄-alkyl or C₁-C₄-alkyl-phenyl; or

together two adjacent OR¹ groups denote —O—CH₂—O—;

R² and R³ independently denote H or C₁-C₄-alkyl; or

NR²R³ denotes —NHC(═O)—NHAr⁵; or

together two adjacent NR²R³ groups denote —NR²—CH—N— or —NR²—CH₂—NR²—;or

together with an adjacent OR¹ group, NR²R³ denotes —NR²—CH₂—O— or—N—CH—O—; or

together with an adjacent SR¹ group, NR²R³ denotes —NR²—CH₂—S— or—N—CH—S—;

Ar⁵ denotes phenyl optionally substituted with R^(h);

R^(h) denotes halogen or C₁-C₄ alkyl;

R⁴ denotes C₁-C₄-alkyl; or

together with an adjacent OR¹ group, R⁴ denotes —CH₂CH₂—O—; or

together two adjacent R⁴ groups denote —(CH)₄ or —(CH₂)₄—;

Y denotes OR⁵ or NR⁶R⁷;

R⁵ denotes H or C₁-C₄-alkyl;

R⁶ and R⁷ independently denote H, C₁-C₈alkyl or C₃-C₆ cycloalkyl.

The present invention also relates to a compound of formula (I′) for usein therapy:

wherein

L¹, L² and L³ independently denote C₁-C₄-alkylene optionally substitutedwith one phenyl;

Ar¹, Ar² and Ar³ independently denote a 5-10 membered heteroaryloptionally substituted with —C(═O)Z or one or more R; or a 6-10 memberedaryl optionally substituted with one or more R;

Z denotes OR^(a) or NR^(b)R^(c);

R^(a) denotes H, C₁-C₄-alkyl, C₃-C₆-cycioalkyl,C₂-C₄-alkylene-O—C₁-C₄-alkyl, C₂-C₄-alkylene-CN; C₁-C₄-alkyl substitutedwith one or two Ar⁴;

R^(b) denotes H, C₃-C₅-cycloalkyl, C₂-C₄-alkylene-CN;C₂-C₄-alkylene-O—C₁-C₄-alkyl; or C₁-C₄-alkyl substituted with one or twoAr⁴;

R^(c) denotes H, C₁-C₈ alkyl, C₃-C₆-cycloalkyl, Ar⁴, C₁-C₄-alkylsubstituted with one or two Ar⁴; OH, O—-C₁-C₄-alkyl,C₂-C₄-alkyl-O—C₁-C₄-alkyl, or C₂-C₄-alkyl-NR^(a)R^(a);

or together NR^(b)R^(c) denotes

wherein X denotes CHZ¹, O, or NZ¹;

Z¹ denotes H, C(═O)H, C(═O)C₁-C₄-alkyl, C(═O)OR^(z), Ar⁴, C₁-C₄-alkyl,C₃-C₆-cycloalkyl, C₁-C₄-alkyl substituted with one or two Ar⁴;C₂-C₄-NR^(d)R^(a), or C₂-C₄-OR¹;

Z² denotes OR^(a) or NR^(g)R^(g);

Ar⁴ denotes 5-6 membered heteroaryl; or phenyl optionally substitutedwith one or more R;

R^(d), R^(e) and R^(f) independently denote H or C₁-C₄-alkyl;

R^(g) denotes R^(b);

R^(z) denotes H, C₁-C₄-alkyl or C₃-C₆-cycloalkyl;

R independently denotes F, Cl, Br, I, C₁-C₄-haloalkyl, OR¹, SR¹, NO₂,NR²R³, R⁴, C(═O)Y, SO₃H, C₁-C₄-alkyl, C₁-C₄-alkyl-phenyl, a 5-memberedheteroaryl; or a phenyl substituted with a 5-membered heteroaryl;

R¹ denotes H, C₁-C₄-alkyl or C₁-C₄-alkyl-phenyl; or

together two adjacent OR¹ groups denote —O—CH₂—O—;

R² and R³ independently denote H or C₁-C₄-alkyl; or

together two adjacent NR²R³ groups denote —NR²—CH—N— or —NR²—CH₂—NR²—;or

together with an adjacent OR¹ group, NR²R³ denotes —NR²—CH₂—O— or—N—CH—O—; or

together with an adjacent SR¹ group, NR²R³ denotes —NR²—CH₂—S— or—N—CH—S—;

R⁴ denotes C₁-C₄-alkyl: or

together with an adjacent OR¹ group, R⁴ denotes —CH₂CH₂—O—; or

together two adjacent R⁴ groups denote —(CH)₄— or —(CH₂)₄—;

Y denotes OR⁵ or NR⁶R⁷;

R⁵ denotes H or C₁-C₄-alkyl; and

R⁶ and R⁷ independently denote H, C₁-C₈-alkyl or C₃-C₆ cycloalkyl.

Preferably, the present invention relates to a compound of formula (Ia)for use in therapy,

wherein

L¹, L² and L³ independently denote C₁-C₄-alkylene optionally substitutedwith one phenyl;

Ar¹, Ar² and Ar³ independently denote a 5-10 membered heteroaryloptionally substituted with —C(═O)Z or one or more R; or a 6-10 memberedaryl optionally substituted with one or more R;

Z denotes OR^(a) or NR^(b)R^(c);

R^(a) denotes H or C₁-C₄-alkyl;

R^(b) denotes H, C₁-C₄ alkyl, C₂-C₄-alkyl-CN; or C₁-C₄-alkyl substitutedwith one or two Ar⁴;

R^(c) denotes H, C₁-C₈ alkyl, C₃-C₆-cycloalkyl, Ar⁴, C₁-C₄-alkylsubstituted with one or two Ar⁴; OH, O—C₁-aralkyl,C₇-C₄-alkyl-O—C₁-C₄-alkyl, or C₂-C₄alkyl-NR^(a)R^(a);

or together NR^(b)R^(c) denotes

wherein X denotes CHZ¹, O, or NZ¹;

Z¹ denotes H, C(═O)H, C(═O)C₁-C₄-alkyl, C(═O)OR^(z), Ar⁴, C₁-C₄-alkylsubstituted with one or two Ar⁴; C₂-C₄—NR^(d)R^(e), or C₂-C₄—OR¹:

Z² denotes OR^(e) or NR^(g)R^(g);

Ar⁴ denotes 5-6 membered heteroaryl; or phenyl optionally substitutedwith one or more R;

R^(d), R^(e) and R^(f) independently denote H or C₁-C₄-alkyl;

R^(g) denotes R^(b);

R^(z) denotes H or C₁-C₄-alkyl;

R independently denotes F, Cl, CF₃, OR¹, NO₂, NR²R³, R⁴, C(═O)Y,C₁-C₄-alkyl, C₁-C₄-alkyl-phenyl, 5-tetrazolyl or

R¹ denotes H, C₁-C₄-alkyl or C₁-C₄-alkyl-phenyl; or

together two adjacent OR¹ groups denote —O—CH₂—O—;

R² and R³ independently denote H or C₁-C₄-alkyl;

R⁴ denotes C₁-C₄-alkyl; or

together with an adjacent OR¹ group, R⁴ denotes —CH₂CH₂—O—;

Y denotes OR⁵ or NR⁶R⁷;

R⁵ denotes H or C₁-C₄-alkyl;

R⁶ and R⁷ independently denote H, C₁-C₈-alkyl or C₃-C₆-cycloalkyl,

The following preferred embodiments relate to the compounds of formula(I), (I′) and (Ia) as defined above:

Preferably, Ar¹, Ar² and Ar³ independently denote a 5-9 memberedheteroaryl optionally substituted with —C(═O)Z or one or more R; or a6-10 membered aryl optionally substituted with one or more R.

Preferably, Ar¹, Ar² and Ar³ independently denote a 5-6 memberedheteroaryl optionally substituted with —C(═O)Z or one or more R; or a6-10 membered aryl optionally substituted with one or more R.

Preferably, Ar¹, Ar² and Ar³ independently denote a 5- or 6-memberedheteroaryl optionally substituted with —C(═O)Z or one or more R; or aphenyl optionally substituted with one or more R.

Preferably, Ar¹, Ar² and Ar³ independently denote a thiazole optionallysubstituted with —C(═O)Z; an oxazole optionally substituted with—C(═O)Z; or a phenyl optionally substituted with one or more R.

Preferably, Ar¹, Ar² and Ar³ independently denote a thiazole optionallysubstituted with —C(═O)Z; or a phenyl optionally substituted with one ormore R.

Preferably, when Ar¹, Ar², Ar³ or Ar⁴ are optionally substituted withone or more R, the (hetero)aryl ring has 0, 1 or 2 substituents.

Preferably, Ar¹ denotes phenyl optionally substituted with one or moreR.

Preferably, Ar² denotes phenyl optionally substituted with one or two R.

Preferably, Ar² denotes phenyl optionally substituted with one or two R;or thiazole optionally substituted with —C(═O)Z.

Preferably, Ar² denotes phenyl optionally substituted with one or two R;

Preferably, Ar² denotes phenyl optionally substituted with one or two R;or

Preferably, L¹, L² and L³ independently denote C₁-C₃-alkylene optionallysubstituted with one phenyl.

Preferably, L¹ denotes —CH₂—.

Preferably, L² denotes —CH₂—.

Preferably, L³ denotes C₁-C₃-alkylene optionally substituted with onephenyl.

Thus, preferred compounds include a compound of formula (II)

wherein

r denotes 0, 1 or 2;

L³ denotes C₁-C₃-alkyl optionally substituted with one phenyl; and

Ar³ and R are as defined above for the compound of formula (I).

Preferably, Ar³ and R in the compound of formula (II) are as defined forthe compound of formula (I′).

Preferably, Ar³ and R in the compound of formula (II) are as defined forthe compound of formula (Ia).

Other preferred compounds include a compound of formula (III)

wherein

r denotes 0, 1 or 2;

L³ denotes C₁-C₃-alkylene optionally substituted with one phenyl; and

Ar³, R and Z are as defined above for the compound of formula (I).

Preferably, Ar³, R and Z in the compound of formula (III) are as definedfor the compound of formula (I′).

Preferably, Ar³, R and Z in the compound of formula (III) are as definedfor the compound of formula (Ia).

The following preferred embodiments relate to the compounds of formula(I), (I′) (Ia), (II) and (III) as defined above, including thoseembodiments of formulae (II) and (III) in which Ar³ and R (and Z in thecase of formula (III)) are as defined for the compound of formula (I′):

Preferably, Ar³ denotes phenyl optionally substituted with one or two R;pyridyl, or 2-furyl.

Preferably, Ar³ denotes phenyl optionally substituted with one or two R;or pyridyl.

Preferably, Ar³ denotes phenyl optionally substituted with one or two R.

Preferably, L³ denotes —CH₂—, —CH(CH₃)— or —CH₂CH₂CH(C₆H₅)—,

Preferably, L³ denotes —CH₂— or —CH₂CH₂CH(C₆H₅)—; and

Ar³ denotes phenyl optionally substituted with one or two R.

Preferably, Z denotes OR^(a),

Preferably, R^(a) denotes H or Me.

Preferably, Z denotes NR^(b)R^(c).

Preferably, R^(b) denotes H or C₁-C₄-alkyl; and

R^(c) denotes H, C₁-C₄ alkyl, cyclopropyl, cyclohexyl, Ar⁴, C₁-C₄-alkylsubstituted with one or two Ar⁴; CH, or O—-C₁-C₄-alkyl;

or together NR^(b)R^(c) denotes

Preferably, R^(b) denotes H or C₁-C₄-alkyl; and

R^(c) denotes H, C₁-C₆ alkyl, C₁-C₄-alkyl substituted with one or twoAr⁴;

or together NR^(b)R^(c) denotes

Preferably, R^(b) denotes H; and

R^(c) denotes H, C₁-C₆ alkyl, C₁-C₄-alkyl substituted with one or twoAr⁴;

or together NR^(b)R^(c) denotes

Preferably, together NR^(b)R^(c) denotes

Preferably, X denotes NZ¹.

Preferably, Z¹ denotes H, C(═O)H, C(═O)C₁-C₄-alkyl; C(═O)OR⁷, Ar⁴,C₁-C₄-alkyl, or C₁-C₄-alkyl substituted with one or two Ar⁴.

Preferably, Z¹ denotes H, C(═O)H, C(═O)C₁-C₄-alkyl, or C(═O)OR⁷.

Preferably, Z¹ denotes H, Ar⁴, C₁-C₄-alkyl, or C₁-C₄-alkyl substitutedwith one or two Ar⁴.

Preferably, Ar⁴ denotes phenyl optionally substituted with one or two R.

Preferably, R^(g) denotes H or C₁-C₄-alkyl.

Preferably, R^(h) denotes F or C₁-C₄-alkyl.

Preferably, R^(h) denotes F or methyl,

Preferably, R^(z) denotes H or methyl.

Preferably, R independently denotes F, Cl, CF₃, OR¹, NO₂, NR²R³, R⁴,C(═O)Y, C₁-C₄-alkyl-phenyl, or

Preferably, R independently denotes F, Cl, CF₃, OR¹, C(═O)Y,—C₄-alkyl-phenyl or

Preferably, R independently denotes F, Cl, CF₃, OR¹, C(═O)Y, C₁-C₄-alkylor C₁-C₄-alkyl-phenyl.

Preferably, Y denotes OR⁵.

Preferably, R⁶ and R⁷ independently denote H and C₁-C₆-alkyl.

The above noted preferred embodiments can of course be combined with oneanother. Thus, particularly preferred embodiments of the inventioninclude:

Embodiment 1

A compound of formula (I) for use in therapy, wherein

L¹, L² and L³ independently denote C₁-C₃-alkylene optionally substitutedwith one phenyl: and

Ar¹ denotes phenyl optionally substituted with one or more R;

Ar² denotes phenyl optionally substituted with one or two R; or thiazoleoptionally substituted with —C(═O)Z;

Ar³ denotes phenyl optionally substituted with one or two R; pyridyl, or2-furyl;

Z denotes OR^(a) or NR^(b)R^(c);

R^(a) denotes H or C₁-C₄-alkyl;

R^(b) denotes H, C₂-C₄-alkyl-CN; or C₁-C₄-alkyl substituted with one ortwo Ar⁴;

R^(c) denotes H, C₁-C₈ alkyl, C₃-C₆ cycloalkyl, Ar⁴, C₁-C₄-alkylsubstituted with one or two Ar⁴; OH, O—C₁-C₄-alkyl,C₂-C₄-alkyl-O—C₁-C₄-alkyl, or C₂-C₄-alkyl-NR^(a)R^(a);

or together NR^(b)R^(c) denotes

wherein X denotes CHZ¹, O, or NZ¹;

Z¹ denotes H, C(═O)H, C(═O)C₁-C₄-alkyl, C(═O)OR^(z), Ar⁴, C₁-C₄-alkylsubstituted with one or two Ar⁴; C₂-C₄-NR^(d)R^(e), or C₂-C₄—OR¹;

Z² denotes OR^(a) or NR^(g)R^(g);

Ar⁴ denotes phenyl optionally substituted with one or two R;

R^(d), R^(e) and R^(f) independently denote H or C₁-C₄-alkyl;

R^(g) denotes R^(b);

R^(z) denotes H or C₁-C₄-alkyl:

R independently denotes F, Cl, CF₃, OR¹, NO₂, NR²R³, R⁴, C(═O)Y,C₁-C₄-alkyl, C₁-C₄-alkyl-phenyl or

R¹ denotes H, C₁-C₄-alkyl or C₁-C₄-alkyl-phenyl; or

together two adjacent OR¹ groups denote —O——CH₂—O—; or

together two adjacent NR²R³ groups denote —NR²—CH—N— or —NR²—CH₂—NR²—;or

together with an adjacent OR¹ group, NR²R³ denotes —NR²—CH₂—O— or—N—CH—O—;

R² and R³ independently denote H or C₁-C₄-alkyl;

R⁴ denotes C₁-C₄-alkyl; or

together with an adjacent OR¹ group, R⁴ denotes —CH₂CH₂—O—;

Y denotes OR⁵ or NR⁶R⁷;

R⁵ denotes H or C₁-C₄-alkyl;

R⁶ and R⁷ independently denote H, C₁-C₃-alkyl or C₃-C₆ cycloalkyl.

Embodiment 2

A compound of formula (I) for use in therapy, wherein

L¹, L² and L³ independently denote C₁-C₃-alkylene optionally substitutedwith one phenyl; and

Ar¹ denotes phenyl optionally substituted with one or more R;

Ar² denotes phenyl optionally substituted with one or two R;

Ar³ denotes phenyl optionally substituted with one or two R; pyridyl, or2-furyl;

Z denotes OR^(a) or NR^(b)R^(c);

R^(a) denotes H or C₁-C₄-alkyl;

R^(b) denotes R^(b) denotes H or C₁-C₄-alkyl; and

R^(c) denotes H, C₁-C₄ alkyl, cyclopropyl, cyclohexyl, Ar⁴, C₁-C₄-alkylsubstituted with one or two Ar⁴; OH, or O—-C₁-C₄-alkyl;

or together NR^(b)R^(c) denotes

X denotes CHZ¹, O, or NZ¹;

Z¹ denotes Z¹ denotes H, C(═O)H, C(═O)C₁-C₄-alkyl, C(═O)OR^(z), Ar⁴,C₁-C₄-alkyl, or C₁-C₄-alkyl substituted with one or two Ar⁴;

Z² denotes OR^(a) or NR^(g)R^(g);

Ar⁴ denotes phenyl optionally substituted with one or two R;

R^(d), R^(e) and R^(f) independently denote H or C₁-C₄-alkyl;

R^(g) denotes R^(b);

R^(z) denotes H or C₁-C₄-alkyl;

R independently denotes F, Cl, CF₃, OR¹, C(═O)Y, C₁-C₄-alkyl,C₁-C₄-alkyl-phenyl or

R¹ denotes H, C₁-C₄-alkyl or C₁-C₄-alkyl-phenyl; or

together two adjacent OR¹ groups denote —O—CH₂—O—;

R² and R³ independently denote H or C₁-C₄-alkyl;

R⁴ denotes C₁-C₄-alkyl; or

together with an adjacent OR¹ group, R⁴ denotes —CH₂CH₂—O—;

Y denotes OR⁵ or NR⁶R⁷;

R⁵ denotes H or C₁-C₄-alkyl:

R⁶ and R⁷ independently denote H and C₁-C₆-alkyl,

Embodiment 3

A compound of formula (I) for use in therapy, wherein

L¹ denotes —CH₂;

L² denotes —CH₂;

L³ denotes —CH₂—, —CH(CH₃)— or —CH₂CH₂CH(C₆H₅)—;

Ar¹ denotes phenyl optionally substituted with one or two R;

Ar² denotes phenyl optionally substituted with one or two R;

Ar³ denotes phenyl optionally substituted with one or two R; pyridyl, or2-furyl;

Z denotes OR^(a) or NR^(b)R^(c):

R^(a) denotes H or C₁-C₄-alkyl;

R^(b) denotes R^(b) denotes H or C₁-C₄-alkyl; and

R^(c) denotes H, C₁-C₄ alkyl, cyclopropyl, cyclohexyl, Ar⁴, C₁-C₄-alkylsubstituted with one or two Ar⁴; OH, or O—C₁-C₄-alkyl;

or together NR^(b)R^(c) denotes

X denotes CHZ¹, O, or NZ¹;

Z¹ denotes Z¹ denotes H, C(═O)H, C(═O)C₁-C₄-alkyl, C(═O)OR^(z), Ar⁴,C₁-C₄-alkyl, or C₁-C₄-alkyl substituted with one or two Ar⁴;

Z² denotes OR^(a) or NR^(g)R^(g):

Ar⁴ denotes phenyl optionally substituted with one or two R;

R^(d), R^(e) and R^(f) independently denote H or C₁-C₄-alkyl;

R^(g) denotes R^(h);

R^(z) denotes H or C₁-C₄-alkyl;

R independently denotes F, Cl, CF₃, OR¹, C(═O)Y, C₁-C₄-alkyl,C₁-C₄-alkyl-phenyl or

R¹ denotes H, C₁-C₄-alkyl or C₁-C₄-alkyl-phenyl; or

together two adjacent OR¹ groups denote —O—CH₂—O—;

R² and R³ independently denote H or C₁-C₄-alkyl;

R⁴ denotes C₁-C₄-alkyl; or

together with an adjacent OR¹ group, R⁴ denotes —CH₂CH₂—O—;

Y denotes OR⁵ or NR⁶R⁷;

R⁵ denotes H or C₁-C₄-alkyl;

R⁶ and R⁷ independently denote H and C₁-C₆-alkyl.

Embodiment 4

A compound of formula (I) for use in therapy, wherein

L¹ denotes —CH₂;

L² denotes —CH₂;

L³ denotes —CH₂—, —CH(CH₃)— or —CH₂CH₂CH(C₆H₅)—;

Ar¹ denotes phenyl optionally substituted with one or two R;

Ar² denotes phenyl optionally substituted with one or two R;

Ar^(a) denotes phenyl optionally substituted with one or two R; pyridyl,or 2-furyl;

Z denotes NR^(b)R^(c);

R^(b) denotes H; and

R^(c) denotes H, C₁-C₆ alkyl, C₁-C₄-alkyl substituted with one or twoAr⁴;

or together NR^(b)R^(c) denotes

X denotes NZ¹;

Z¹ denotes Z¹ denotes H, C(═O)H, C(═O)C₁-C₄-alkyl, C(═O)OR⁷, Ar⁴,C₁-C₄-alkyl, or C₁-C₄-alkyl substituted with one or two Ar⁴;

denotes OR^(a) or NR^(g)R^(g);

Ar⁴ denotes phenyl optionally substituted with one or two R;

R^(d), R^(e) and R^(f) independently denote H or C₁-C₄-alkyl;

R^(g) denotes R^(b);

R^(z) denotes H or C₁-C₄-alkyl;

R independently denotes F, Cl, CF₃, OR¹, C(═O)Y, C₁-C₄-alkyl,C₁-C₄-alkyl-phenyl or

R¹ denotes H, C₁-C₄-alkyl or C₁-C₄-alkyl-phenyl; or

together two adjacent OR¹ groups denote —O—CH₂—O—;

R² and R³ independently denote H or C₁-C₄-alkyl;

R⁴ denotes C₁-C₄-alkyl; or

together with an adjacent OR¹ group, R⁴ denotes —CH₂CH₂—O—;

Y denotes OR⁵ or NR⁶R⁷;

R^(b) denotes H or C₁-C₄-alkyl;

R⁶ and R⁷ independently denote H and C₁-C₆-alkyl,

Embodiment 5

A compound of formula (I) for use in therapy, wherein

L¹ denotes —CH₂;

L² denotes —CH₂;

L³ denotes —CH₂—, —CH(CH₃)— or —CH₂CH₂CH(C₆H₅)—;

Ar¹ denotes phenyl optionally substituted with one or two R;

Ar² denotes phenyl optionally substituted with one or two R;

Ar^(a) denotes phenyl optionally substituted with one or two R; pyridyl,or 2-furyl;

Z denotes NR^(b)R^(c);

R^(b) denotes H; and

R^(c) denotes H, C₁-C₆ alkyl, C₁-C₄-alkyl substituted with one or twoAr⁴;

or together NR^(b)R^(c) denotes

X denotes NZ¹;

Z¹ denotes Z¹ denotes H, C(═O)H, C(═O)C₁-C₄-alkyl, C(═O)OR^(z), Ar⁴,C₁-C₄-alkyl, or C₁-C₄-alkyl substituted with one or two Ar⁴;

Z² denotes OR^(a) or NR^(g)R^(g);

Ar⁴ denotes phenyl optionally substituted with one or two R;

R^(d), R^(e) and R^(f) independently denote H or C₁-C₄-alkyl;

R^(g) denotes R^(b);

R^(z) denotes H or C₁-C₄-alkyl;

R independently denotes F, Cl, CF₃, OR¹, C(═O)Y, C₁-C₄-alkyl orC₁-C₄-alkyl-phenyl;

R¹ denotes H, C₁-C₄-alkyl or C₁-C₄-alkyl-phenyl; or

together two adjacent OR¹ groups denote —O—CH₂—O—;

R² and R³ independently denote H or C₁-C₄-alkyl;

R⁴ denotes C₁-C₄-alkyl; or

together with an adjacent OR¹ group, R⁴ denotes —CH₂CH₂—O—;

Y denotes OR⁵ or NR⁶R⁷;

R⁵ denotes H or C₁-C₄-alkyl;

R⁶ and R⁷ independently denote H and C₁-C₆-alkyl.

Typically, the compound of formula (I) (or formula (I′)) contains atleast one group capable of acting as a hydrogen bond donor, and/or atleast one group capable of acting as a hydrogen bond acceptor.

Preferably, the compound of formula (I) (or formula (I′)) will containat least one, more preferably two, groups capable of acting as ahydrogen bond donor.

Preferably, the compound of formula (I) (or formula (I′)) will containat least one, more preferably two, groups capable of acting as ahydrogen bond acceptor.

By “hydrogen bond donor” is meant a group containing a hydrogen atomcapable of forming a hydrogen bond, such as OH or NH. Hydrogen bonddonors may also form part of a ring, such as tetrazole NH groups.

By “hydrogen bond acceptor” is meant a group capable of forming ahydrogen bond with a hydrogen atom, such as OMe or NMe₂. Hydrogen bondacceptors may also form part of a ring, such as pyridyl nitrogen atoms.

In some embodiments, the L¹Ar¹ and L²Ar² moiety in the compound offormula (I) (or formula (I′)) are identical.

In some embodiments, L¹Ar¹, L²Ar² and L³Ar³ in the compound of formula(I) (or formula (I′)) are all different.

In some embodiments, the compound of formula (I) (or formula (I′)) doesnot have the following structure (i.e. in some embodiments the compoundshaving the following structure are excluded from the definition of anygeneric formula which may encompass them and do not form part of theclaimed invention):

ethyl1-[2-(dibenzylamino)ethyl]-4-(4-fluorobenzyl)-3-methyl-1H-pyrazole-5-carboxylate

ethyl1-[2-(dibenzylamino)ethyl]-4-(4-fluorobenzyl)-5-methyl-H-pyrazole-3-carboxylate

2-[({[4-(benzyloxy)phenyl]methyl}[3-fluorophenyl)methyl]amino)methyl)-1,3-thiazole-4-carboxylicacid

2-[({4-(benzyloxy)phenyl]methyl}(2,2-diphenylethyl)amino)methyl]-1,3-thiazole-4-carboxylicadd

2-[(dibenzylamino)methyl]-1,3-thiazole-4-carboxylic add

ethyl2-[({[4-(benzyloxy)phenyl]methyl}[(4-fluorophenyl)methyl]amino)methyl-1,3-thiazole-4-carboxylate

2-[({[4-(benzyloxy)phenyl]methyl}(pyridin-4-ylmethyl)amino)methyl]-1,3-thiazole-4-carboxylicacid

2-[(}[4-(benzyloxy)phenyl]methyl}(pyridin-3-ylmethyl)amino)methyl]-1,3-thiazole-4-carboxylicacid

2-({[(4-fluorophenyl)methyl][(4-biphenyl)methyl]amino}methyl)-1,3-thiazole-4-carboxylicacid

ethyl2-[({[4-(benzyloxy)phenyl]methyl}(pyridin-3-ylmethyl)amino)methyl]-1,3-thiazole-4-carboxylate

ethyl2-[({[4-(benzyloxy)phenyl]methyl}(pyridin-4-ylmethyl)amino)methyl]-1,3-thiazole-4-carboxylate

2-{[(4-benzyloxy-benzyl)-(4-fluoro-benzyl)-amino]methyl}-thiazole-4-carboxylicacid ethylamide

{[4-(benzyloxy)phenyl]methyl}[4-fluorophenyl)methyl]{1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]methyl}amine

{[4-(benzyloxy)phenyl]methyl}[4-fluorophanyl)methyl[{4-(trifluoromethyl)-1H-imidazol-2-yl]methyl}amine

2-[({[4-(benzyloxy)phenyl]methyl}[4-fluorophenyl)methyl]amino)methyl]-1H-imidazole-4-carboxylicacid

2-{[biphenyl-4-ylmethyl-(4-fluoro-benzyl)-amino]-methyl}-thiazole-4-carboxylicacid ethylester

Preferred compounds according to formula have the following structure:

Particularly preferred compounds according to formula (I) have thefollowing structure:

The invention relates to compounds of formula (I) for use in therapy.However, some of the compounds themselves are previously unknown. Theinvention therefore also provides novel compounds that find use intherapy, for example compounds of formula (I) having the followingstructure:

Preferred novel compounds of formula (I) for use in therapy are:

It is to be understood that where the preferred embodiments mentionedabove are not mutually exclusive, they can be combined with one another.For example, the skilled person would understand that the abovepreferred embodiments in which Ar¹ (e.g. Ar¹) denotes phenyl optionallysubstituted with one or more R can be combined with the preferredembodiments in which R denotes independently denotes F, Cl, CF₃, OR¹,C(═O)Y, C₁-C₄-alkyl or C₁-C₄-alkyl-phenyl. The same holds true for theother non-mutually exclusive preferred embodiments mentioned above. Theskilled person would understand which embodiments where mutuallyexclusive and would thus readily be able to determine the combinationsof preferred embodiments that are contemplated by the presentapplication.

As used herein, “alkyl” or “alkylene” is intended to include bothbranched and straight-chain saturated aliphatic hydrocarbon groupshaving the specified number of carbon atoms. For example, “C₁-C₆ alkyl”is intended to include C₁, C₂, C₃, C₄, C₅ and C₆ alkyl groups. Examplesof alkyl include, but are not limited to, methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl,2-methylbutyl, 2-methylpentyl, 2-ethylbutyl, 3-methylpentyl, and4-methylpentyl.

By “haloalkyl” is meant both branched and straight chain saturatedaliphatic hydrocarbon groups having the specified number of carbonatoms, wherein at least one of the hydrogen atoms has been replaced byF, Cl, Br or I. Preferably, haloalkyl refers to perfluoralkyl.

The term “cycloalkyl” refers to cyclized alkyl groups, including mono-,bi- or poly-cyclic ring systems. Example cycloalkyl groups include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,norbornyl, adamantyl and the like. Branched cycloalkyl groups such as1-methylcyclopropyl and 2-methylcyclopropyl are included in thedefinition of “cycloalkyl”.

As used herein, the term “aryl”, is intended to mean an aromatic moietycontaining, if specified, the specified number of carbon atoms; forexample phenyl or naphthyl.

As used herein, the term “heteroaryl”, is intended to mean an aromaticmoiety containing, if specified, the specified number of atoms with atleast one of the ring atoms being selected from N, O or S. Examples ofheteroaryl rings include pyrrole, furan, thiophene, imidazole, pyrazole,1,2,3-triazole. 1,2,4-triazole, tetrazole, thiazole, oxazole,isooxazole, benzofuran, isobenzofuran, indole, isoindole,benzothiophene, benzo[c]thiophene, benzinimdazole, indazole, purine,benzoxazole, benzothiazole, pyridine, pyrimidine, pyrazine, pyrazidine,quinoline, isoquinoline, quinoxaline, quinazoline and cinnoline.

The compounds of formula (I), (I′), (Ia), (II) and (III) are found toselectively target and disrupt Phospholamban-AKAP18δ, indicating thatthey would find use as cardioprotective agents following myocardialinfarction.

Therefore, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in therapy.

As mentioned above, these pharmaceutical compositions may be used fortreating or preventing conditions in which PKA type II signalling whichregulates SERCA2 is abnormal, in particular when the activity of thispathway is elevated or reduced. Furthermore, anchoring disruption may bebeneficial also when the signalling is normal in cases when the heart isdamaged and needs to be protected from adrenergic stimuli and pacing.

Thus, viewed from a further aspect, the present invention preferablyprovides a method of regulating SERCA2 activity in a human or non-humananimal wherein a pharmaceutical composition as described hereinbefore isadministered to said animal.

Viewed another way, the present invention provides a method of treatingor preventing diseases or disorders exhibiting abnormal SERCA2 activityor which would benefit from a reduction or elevation in the activity ofSERCA2 in a human or non-human animal wherein a pharmaceuticalcomposition as described hereinbefore is administered to said animal.

Alternatively stated, the present invention provides the use of apharmaceutical composition as defined herein for the preparation of amedicament for the regulation of SERCA2 activity, preferably asdescribed hereinbefore.

Alternatively stated, the present invention provides the use of apharmaceutical composition as defined herein for the preparation of amedicament for treating or preventing diseases or disorders exhibitingabnormal SERCA2 activity or which would benefit from a reduction orelevation in the activity of SERCA2.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in treating or preventing diseases ordisorders exhibiting abnormal SERCA2 activity or which would benefitfrom a reduction or elevation in the activity of SERCA2.

A number of the examples show that the regulation of SERCA2 activityprovided by the compounds of the invention is associated with theireffect on PKA type II signalling.

Thus, viewed from a further aspect the present invention preferablyprovides a method of treating or preventing diseases or disordersexhibiting abnormal PKA type II signalling that regulates SERCA2activity or which would benefit from a reduction or elevation in thelevels of SERCA2 mediated PKA type II signalling, preferably asdescribed hereinbefore, in a human or non-human animal wherein apharmaceutical composition as described hereinbefore is administered tosaid animal.

Alternatively stated, the present invention provides the use of apharmaceutical composition as defined herein for the preparation of amedicament for the treatment or prevention of diseases or disordersexhibiting abnormal PKA type II signalling that regulates SERCA2activity or which would benefit from a reduction or elevation in thelevels of SERCA2 mediated PKA type II signalling, preferably asdescribed hereinbefore.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in treating or preventingcardiovascular disease.

As referred to herein “cardiovascular disease” refers to a disease ordisorder of the heart or vascular system which may be congenital oracquired and encompasses diseases such as congenital heart failure,hypertension, myocardial infarction, congestive heart failure,reperfusion damage, dilated cardiomyopathy, post infarction heartfailure, arrhythmia, atherosclerotic peripheral arterial disease andalveolar hypoxia leading to pulmonary hypertension and right ventriclefailure.

As used herein, “treating” refers to the reduction, alleviation orelimination, preferably to normal levels, of one or more of the symptomsof said disease, disorder or condition which is being treated, e.g.normal blood pressure, cardiac function, etc., relative to the symptomsprior to treatment. Where not explicitly stated, treatment encompassesprevention. “Preventing” refers to absolute prevention, i.e. maintenanceof normal levels with reference to the extent or appearance of aparticular symptom (e.g. hypertension) or reduction or alleviation ofthe extent or timing (e.g. delaying) of the onset of that symptom.

Preferably, the present invention relates to a compound of formula,(I′), (Ia), (II) or (III) for use in treating conditions related to theheart.

By “conditions related to the heart” is meant and disease or disorderrelated of the heart which may be congenital or acquired and encompassesdiseases such as congenital heart failure, myocardial infarction, postinfarction heart failure, congestive heart failure, reperfusion damage,dilated cardiomyopathy and arrhythmia.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in treating myocardial infarction.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in treating congestive heart failure.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in protecting against reperfusiondamage.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in protecting against post infarctionheart failure.

Preferably, the present invention relates to a compound of formula),(I′), (Ia), (II) or (III) for use in protecting against congenital heartfailure.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in protecting against dilatedcardiomyopathy.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in protecting against arrhythmia heartfailure.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in interfering with A-kinase proteins.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in inhibiting phospholambanphosphorylation.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in inhibiting PKA phospholambanphosphorylation upon adrenergic stimulation.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in inhibiting PKA-mediatedphospholamban phosphorylation upon adrenergic stimulation.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in regulating SERCA2 activity.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in mediating adrenergic pacing of theheart.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in regulating the adrenergic pacing ofthe heart.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in preventing arrhythmia.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in inhibiting protein kinase A bindingto A-kinase anchor proteins.

Preferably, the present invention relates to a compound of formula (I),(I′), (Ia), (II) or (III) for use in inhibiting AKAP18δ binding tophospholamban.

The present invention also relates to methods oftreating/inhibiting/protecting against/mediating (as the case may be)the above conditions, comprising administering to a human or non-humananimal (e.g. a mammal) in need thereof a compound according to formula(I), (I′), (II), or (III).

Subjects which may be treated are preferably mammalian, preferablyhumans and companion or agricultural animals such as dogs, cats,monkeys, horses, sheep, goats, cows, rabbits, rats and mice.

Preferred mammals are humans.

The present invention also relates to the use of a compound according toformula (I), (I′), (II), or (III) for treating/inhibiting/protectingagainst/mediating (as the case may be) the above conditions.

The present invention also relates to a compound according to formula(I), (I′), (II), or (III) for use in the manufacture of a medicament foruse in treatinglinhibiting/protecting against/mediating (as the case maybe) the above conditions.

As shown by the examples below, the compounds of the invention arecapable of influencing phospholamban phosphorylation. This is believedto be the first time this the activity of this target has been modulatedby chemical moieties.

Thus, viewed from a further aspect the present invention preferablyprovides a method of treating or preventing diseases or disordersassociated with phospholamban phosphorylation, comprising administeringto a subject in need thereof a pharmaceutically effective amount of acompound with molecular weight below 1000 Daltons (or g/mol) orpharmaceutically acceptable salts thereof.

By “diseases or disorders associated with phospholamban phosphorylation”is meant conditions which arise due to abnormal phospholambanphosphorylation, such as but not limited to post infarction heartfailure and congestive heart failure, as well as conditions that mayoccur in patients showing normal phospholamban phosphorylation but whichwould benefit from a cardioprotective effect due to inhibition ofphospholamban phosphorylation, such as but not limited to congenitalheart failure, myocardial infarction, reperfusion damage, dilatedcardiomyopathy, and arrhythmia.

Preferred diseases or disorders associated with phospholambanphosphorylation are cardiovascular diseases.

Preferred diseases or disorders associated with phospholambanphosphorylation are cardiovascular diseases selected from congenitalheart failure, myocardial infarction, post infarction heart failure,congestive heart failure, reperfusion damage, dilated cardiomyopathy andarrhythmia.

The invention also relates to a method of regulating phospholambanphosphorylation comprising administering to a subject a pharmaceuticallyeffective amount of a compound with molecular weight below 1000 Daltons,or pharmaceutically acceptable salts thereof.

The invention also relates to a method of inhibiting phospholambanphosphorylation comprising administering to a subject a pharmaceuticallyeffective amount of a compound with molecular weight below 1000 Daltons,or pharmaceutically acceptable salts thereof.

In preferred embodiments, these methods use a nitrogen-containingcompound. More preferably, said nitrogen-containing compound is anamine. Even more preferably, said amine is a tertiary amine.

Most preferably, said amine is an alkylaryl or alkylheteroaryl tertiaryamine.

Thus, in a preferred aspect, the invention relates to a method oftreating or preventing diseases or disorders associated withphospholarnban phosphorylation, comprising administering to a subject inneed thereof a pharmaceutically effective amount of a alkylaryl oralkylheteroaryl tertiary amine with molecular weight below 1000 Daltonsor pharmaceutically acceptable salts thereof.

In such embodiments, “alkylaryl” means an alkylene group bonded to anaromatic ring containing only carbon atoms, which may optionally besubstituted. By “alkylheteroaryl” is meant an alkylene group bonded toan aromatic ring containing carbon atoms and at least one heteroatom,which may optionally be substituted.

In preferred embodiments, the alkylaryl or alkylheteroaryl tertiaryamine is a compound of formula (I), (I′), (Ia), (II) or (III).

The present invention also relates to pharmaceutical compositionscomprising a compound according to formula (I), (I′), (Ia), (II), or(III) and one or more pharmaceutically acceptable excipients and/ordiluents.

By “pharmaceutically acceptable” is meant that the ingredient must becompatible with other ingredients in the composition as well asphysiologically acceptable to the recipient.

Pharmaceutical compositions for use according to the invention may beformulated in conventional manner using readily available ingredients.Thus, the active ingredient may be incorporated, optionally togetherwith other active substances as a combined preparation, with one or moreconventional carriers, diluents and/or excipients, to produceconventional galenic preparations such as tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions (as injection orinfusion fluids), emulsions, solutions, syrups, aerosols (as a solid orin a liquid medium), ointments, soft and hard gelatin capsules,suppositories, sterile injectable solutions, sterile packaged powders,and the like. Biodegradable polymers (such as polyesters,polyanhydrides, polylactic acid, or polyglycolic acid) may also be usedfor solid implants. The compositions may be stabilized by use offreeze-drying, undercooling or Permazyme.

Suitable excipients, carriers or diluents are lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,calcium carbonate, calcium lactose, corn starch, aglinates, tragacanth,gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol,water/glycol, water/polyethylene, glycol, propylene glycol, methylcellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc,magnesium stearate, mineral oil or fatty substances such as hard fat orsuitable mixtures thereof. Agents for obtaining sustained releaseformulations, such as carboxypolymethylene, carboxymethyl cellulose,cellulose acetate phthalate, or polyvinylacetate may also be used. Thecompositions may additionally include lubricating agents, wettingagents, viscosity increasing agents, colouring agents, granulatingagents, disintegrating agents, binding agents, osmotic active agents,emulsifying agents, suspending agents, preserving agents, sweeteningagents, flavouring agents, adsorption enhancers, e.g. for nasal delivery(bile salts, lecithins, surfactants, fatty acids, chelators) and thelike. The compositions of the invention may be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration of the patient by employing procedures well knownin the art.

The active ingredient in such compositions may comprise from about 0.01%to about 99% by weight of the formulation, preferably from about 0.1 toabout 50%, for example 10%.

The invention also extends to pharmaceutical compositions as describedabove for use as a medicament.

It will be understood that preferred methods/uses/compositions describedabove utilise the preferred compounds of formula (I), (I′), (Ia), (II)and (III) defined above, particularly the specific compounds mentionedabove.

The administration may be by any suitable method known in the medicinalarts, including for example oral, parenteral (e.g. intramuscular,subcutaneous, intraperitoneal or intravenous) percutaneous, buccal,rectal or topical administration or administration by inhalation.

The preferred routes of administration are oral administration andintravenous administration. Preferred formulations for the compositionof the invention are therefore as a tablet, a capsule or an intravenoussolution.

The tablets are typically prepared by direct compression or agranulation procedure, for example using standard fluid bed technology.The tablets are preferably coated with film coating or another coatingsuch as an enteric coating.

The capsules are preferably gelatine capsules.

The composition for injection can be a ready to use solution or a drymaterial to be dissolved before administration. All intravenouscompositions are sterile. Any sterilization method may be used, such asheat sterilization and aseptic preparation.

The compositions of the invention may contain the compound of formula(I) (or formula (I′)) as a physiologically acceptable salt thereof. Suchsalts might typically be HCl, HBr, sulphate salt, phosphate salt,nitrate salt and salts with sulphonic acids like for example methanesulphonic acid. Other salts include organic salts like acetate, citrateand fumarate. The salt form of the compound of formula (I) (or formula(I′)) is preferred for intravenous administration.

The unit dose will vary depending upon choice of compound and disease ordisorder being treated.

Typically, the unit dose will vary from 0.1 mg to 500 mg; morepreferably from 1 mg to 300 mg. A typical daily dose will be from 0.1 mgto 2 grams, more preferably 1 mg to 1 g, even more preferably 1 mg to600 mg.

Typical daily doses per kg body weight of the patient vary from 5mg/kg/day to 50 mg/kg/day, preferably from 10 mg/kg/day to 40 mg/kg/day.

Typically, the tablet or capsule weight is between 70 mg and 1 gram.Typically, the injection or infusion volume is between 0.3 ml and 500ml.

The dosing regime will vary depending upon the clinical situation.Typical average dosing will be once, twice or three times a day,preferably once or twice a day.

The precise dosage of the active compound to be administered and thelength of the course of treatment will of course, depend on a number offactors including for example, the age and weight of the patient, thespecific condition requiring treatment and its severity, and the routeof administration.

The compounds of formula (I) (such as those of formula (I′)) typicallyshow an EC₅₀ value (as determined by the AlphaScreen described in theExamples) of 200 μM or below, preferably 100 μM or below, morepreferably 50 μM or below, more preferably 25 μM or below, mostpreferably 10 μM or below, such as 5 μM or below.

The method of treatment according to the invention may advantageously becombined with administration of one or more active ingredients which areeffective in treating the disorder or disease to be treated. Thus,pharmaceutical compositions of the invention may additionally containone or more of such active ingredients.

Co-administration with other cardiovascular drugs is particularlypreferred. For example, it is preferable to co-administer the compoundsof formula (I) (or formula (I′)) with drugs that treat hypertension,heart failure, arrhythmia and post infarction. It is more preferable toco-administer the compounds of formula (I) (or formula (I′)) with drugsthat treat hypertension, heart failure, arrhythmia and post infarctionmyocardial reperfusion syndrome.

The most preferred drugs to be administered together with the compoundsof formula (I) or (I′) are beta-blockers, calcium antagonists,ACE-inhibitors, ATII/-blockers and anti-arrhythmic drugs.

The following Examples are given by way of illustration only in whichthe Figures referred to are as follows:

FIG. 1: Set-up of GST-AKAP18δ: PLB-biotin interaction assay byAlphaScreen (A) and cross-titration of the preparations of GST-AKAP18δand biotinylated PLB (B)

FIG. 2: Concentration--response curves of compound 2b in the AKAP18δ-PLBAlphaScreen assay. Compound concentration (x-axis) is logarithmic.

FIG. 3: Concentration-response curves of compound 2g in the AKAP18δ-PLBAlphaScreen assay. Compound concentration (x-axis) is logarithmic.

FIG. 4: Concentration-response curves of compound 3b in the AKAP18δ-PLBAlphaScreen assay. Compound concentration (x-axis) is logarithmic.

FIG. 5: Concentration-response curves of compound 3c in the AKAP18δ-PLBAlphaScreen assay. Compound concentration (x-axis) is logarithmic.

FIG. 6: Concentration-response curves of compound 3d in the AKAP18δ-PLBAlphaScreen assay. Compound concentration (x-axis) is logarithmic.

FIG. 7: Disruption of the AKAP18δ-PLB complex influences PLB-Ser¹⁶phosphorylation. Rat neonatal cardiomyocytes treated with compounds 24hours prior stimulation with isoproterenol (100 nM, 5 minutes). Thehistogram shows levels of phosphorylated Ser¹⁶-PLB quantified bydensiometery relative to actin levels.

FIG. 8: Percent viability after 24 hours incubation with synthesizedcompounds.

FIG. 9: Disruption of the AKAP18δ-PLB complex influences PLB-Ser¹⁶phosphorylation. Rat neonatal cardiomyocytes treated with compound 2g(A) or 3d (C) 24 hours prior stimulation with isoproterenol (100 nM, 5minutes). Rat adult cardiomyocytes treated with compound 2g (B) 1 hourprior to stimulation with isoproterenol (10 nM, 5 minutes). Thehistograms show levels of phosphorylated Ser¹⁶-PLB quantified bydensitometry on Western blots (relative to actin levels).

FIG. 10: Compound 2g regulates SERCA2-activity. A novel patch clampbased technique was performed to directly assess SERCA-activityindependently of receptor stimulation in intact cardiomyocytes. Bydialyzing cardiomyocytes with a fixed level of cAMP with and withoutpresence of compound 2g, preliminary data indicates ability of compound2g to regulate SERCA2-activity in intact adult cardiomyocytes.

EXAMPLES

Binding Assay

Stable and optimal assay conditions were determined by cross-titratingGST-AKAP18 δ and biotinylated PLB using 10 μg/ml glutathione acceptorbeads and 10 μg/ml streptavidin donor beads in an AlphaScreen assay. Theexcitation wavelength was 680 nm, with the emission wavelength being520-560 nm. Signal intensity in each well was registered and the optimalconcentration to use was chosen to be the concentration prior to thepeak of the signal. The set up of the assay and cross-titration resultsare shown in FIGS. 1A and 1B.

Concentrations to give a reliable signal were determined to be between 2and 16 nM for AKAP18δ-GST and 4 and 20 nM PLB-biotin for relevantpreparations of protein, for example 4 nM and 20 nM, respectively.

Example 1 Screening

A compound library consisting of 79,000 different small molecularcompounds was screened with the above assay. The following compounds offormulae (l) were identified as having relatively low EC₅₀ values (2.0μM-137 μM).

TABLE 1 Group of compounds of formulae (I) Structure EC₅₀

41.6 μM

137 μM

10.6 μM

18 μM

21.1 μM

36.4 μM

39.5 μM

60.5 μM

94.3 μM

98.3 μM

25.6 μM

35.4 μM

29.6 μM

3.8 μM

215.9 μM

7.5 μM

9.3 μM

3.6 μM

2.9 μM

22.2 μM

2.5 μM

5.2 μM

2.0 μM

SYNTHETIC EXAMPLES Synthesis of Intermediate 1 Preparation ofN-(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine

To a round bottom flask with a magnetic stirrer bar 4-picolylamine (0.43g, 4 mmol) and 4-fluorobenzaldehyde (0.63 g, 5 mmol) were added togetherwith 10 m methanol, NaCNBH₃ (0.32 g, 5 mmol) and a few granules of 4 Åmolecular sieve. The solution was stirred overnight at room temperatureand the progress of the reaction was monitored by thin layerchromatography (TLC). After filtration the solution was concentrated invacua and the resulting liquid was diluted with 30 ml dichloromethaneand extracted with 3×30 ml 0.05 M HCl. The combined aqueous layer wasmade basic with NaOH until the pH 10-11 and the solution was extractedwith 2×100 ml dichloromethane. The organic layers were combined anddried over anhydrous MgSO₄, filtered and evaporated to giveN-(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine as a pale yellow oil.Yield: 0.66 g (76%). ¹H NMR (300 MHz, CDCl₃): δ 8.54 (2H, d), 7.30 (4H,m), 7.01 (2H, m), 3.81 (2H, s), 3.77 (2H, s).

Synthesis of Intermediate 2 Preparation ofN-(4-(tert-butyl)benzyl)-1-(pyridin-3-yl)methanamine

To a round bottom flask with a magnetic stirrer bar 3-picolylamine (0.43g, 4 mmol) and 4-tert-butylbenzaldehyde (0.81 g, 5 mmol) were addedtogether with 10 ml methanol, NaCNBH₃ (0.32 g, 5 mmol) and a fewgranules of 4 Å molecular sieve. The solution was stirred for 2-12 hoursat room temperature and the progress of the reaction was monitored bythin layer chromatography (TLC). After filtration the solution wasconcentrated in vacuo and the resulting liquid was diluted with 30 mldichloromethane and extracted with 3×30 ml 0.05 M HCl. The combinedaqueous layer was made basic with NaOH until the pH 10-11 and thesolution was extracted with 2×100 ml dichloromethane. The organic layerswere combined and dried over anhydrous MgSO₄, filtered and evaporated togive N-(4-(tert-butyl)benzyl)-1-(pyridin-3-yl)methanamine as a paleyellow oil. Yield: 0.61 g (60%). ¹H NMR (300 MHz, CDCl₃): δ 8.59 (1H,d), 8.53 (1H, d), 7.78 (1H, d), 7.39 (2H, d), 7.29 (3H, m), 3.86 (2H,s), 3.81 (2H, s), 1.33 (9H, s). ¹³C NMR (75 MHz, CDCL₃): δ 150.5, 150,148.9, 136.2, 135.9, 134.8, 128.2, 125.6, 123.6, 52.5, 50.1, 34.6, 31.5.

Synthesis of Intermediate 3 Preparation of2-(((pyridin-4-ylmethyl)amino)methyl)benzene-1,4-diol)

To a round bottom flask with a magnetic stirrer bar 4-picolylamine (0.43g, 4 mmol) and 2,5-dihydroxybenzaldehyde (0.69 g, 5 mmol) were addedtogether with 10 ml methanol, NaCNBH₃ (0.32 g, 5 mmol) and a fewgranules of 4 Å molecular sieve. The solution was stirred for 2-12 hoursat room temperature and the progress of the reaction was monitored bythin layer chromatography (TLC). After filtration the solution wasconcentrated in vacuo and the resulting liquid was diluted with 50 mldichloromethane and extracted with 3×50 ml 0.05 M HCl. The combinedaqueous layer was made basic with NaOH until the pH was 10 and thesolution was extracted with 2×150 ml dichloromethane. The organic layerswere combined and dried over anhydrous MgSO₄, filtered and evaporated togive 2-(((pyridin-4-ylmethyl)amino)methyl)-benzene-1,4-diol as a highlyviscous brown oil. Yield: 0.26 g (28%). ¹H NMR (300 MHz, DMSO): δ 8.51(2H, m), 7.34 (2H, d), 6.51 (2H, m). ¹³C NMR (75 MHz, DMSO): δ 149.3,149.2, 148.8, 148.5, 125, 122.7, 115.2, 113.7, 78.8, 50.4, 48.7.

Synthesis of Intermediate 4 Preparation ofN,N-dimethyl-4-(((pyridin-4-ylmethyl)amino)methyl)aniline

To a round bottom flask with a magnetic stirrer bar 4-picolylamine (0.43g, 4 mmol) and 4-(dimethylamino)benzaldehyde (1.34 g, 9 mmol) were addedtogether with 10 ml methanol, NaCNBH₃ (0.57 g, 9 mmol) and a fewgranules of 4 Å molecular sieve. The solution was stirred for 2-12 hoursat room temperature and the progress of the reaction was monitored bythin layer chromatography (TLC). After filtration the solution wasconcentrated in vacuo and the resulting liquid was diluted with 50 mldichloromethane and extracted with 3×50 ml 0.05 M HCl. The combinedaqueous layer was made basic with NaOH until the pH was 10 and thesolution was extracted with 2×150 ml dichloromethane. The organic layerswere combined and dried over anhydrous MgSO₄, filtered and evaporated togive N,N-dimethyl-4-(((pyridin-4-ylmethyl)amino)methyl)aniline as a darkbrown oil. Yield: 0.85 g (88%),

¹H NMR (300 MHz, CDCl₃): δ 8,56 (2H, d), 7.25 (4H, m), 6.74 (2, d), 3.82(2H, s), 3.73 (2H, s), 2.96 (6H, s). ¹³C NMR (75 MHz, CDCl₃): δ 150.1,149.9, 149.7, 129.2, 128.7, 127.7, 123.2, 112.8, 52.8, 51.7, 40.8.

Synthesis of Intermediate 5 Preparation of methyl4-((benzylamino)methyl)benzoate

To a round bottom flask with a magnetic stirrer bar benzylamine (0.215g, 2 mmol) and methyl 3-formylbenzoate (0.41 g, 2.5 mmol) were addedtogether with 10 ml methanol, NaCNBH₃ (0.16 g, 2.5 mmol) and a fewgranules of 4 Å molecular sieve, The solution was stirred for 2-12 hoursat room temperature and the progress of the reaction was monitored bythin layer chromatography (TLC). After filtration the solution wasconcentrated in vacuo and the resulting liquid was diluted with 50 mldichloromethane and extracted with 3×50 ml 0.05 M HCl. The combinedaqueous layer was made basic with NaOH until the pH 10-11 and thesolution was extracted with 2×150 ml dichloromethane. The organic layerswere combined and dried over anhydrous MgSO₄, filtered and evaporated togive methyl 4-((benzylamino)methyl)benzoate as a light brown oil. Yield:0.37 g (36%). ¹H NMR (300 MHz. CDCl₃): δ 8.00 (2H, m), 7.59 (1H, d),7.36 (6H, m), 3.94 (3H, s), 3.87 (2H, s), 3.83 (2H, s) ¹³C NMR (75 MHz,CDCl₃): δ 167.3, 140.9, 140.3, 132.9, 130.4, 129.4, 128.6, 128.4, 128.3,127.2, 53.4, 52.9, 52.2.

Synthesis of Intermediate 6 Preparation of4-((benzylamino)methyl)-N,N-dimethylaniline

To a round bottom flask with a magnetic stirrer bar benzylamine (1.07 g,10 mmol) and 4-(dimethylamino)benzaldehyde (1.79 g, 12 mmol) were addedtogether with 10 ml methanol, NaCNBH₃ (0.76 g, 12 mmol) and a fewgranules of 4 Å molecular sieve. The solution was stirred for 3 hours atroom temperature and the progress of the reaction was monitored by thinlayer chromatography (TLC). After filtration the mixture wasconcentrated in vacuo and 300 mg of the crude product was purified byflash chromatography using ethyl acetate/CH₂Cl₂ (1:9) giving4-((benzylamino)methyl)-N,N-dimethylaniline as a yellow/brown oil. ¹HNMR (300 MHz, CDCl₃): δ 7.34 (5H, m), 7.19 (2H, d), 6.68 (2H, d), 3.81(2H, s), 3.75 (2H, s), 2.92 (6H, s).

Synthesis of Intermediate 7 Preparation ofN-(4-methoxybenzyl)-1-(pyridin-4-yl)methanamine

To a round bottom flask with a magnetic stirrer bar, 4-picolylamine(0.43 g, 4 mmol) and 4-methoxybenzaldehyde (1.22 g, 9 mmol) were addedwith 10 ml methanol, NaCNBH₃ (0.57 g, 9 mmol) and a few granules of 4 Åmolecular sieve. The solution was stirred for 2-12 hours at roomtemperature and the progress of the reaction was monitored by thin layerchromatography (TLC). After filtration the solution was concentrated invacuo and to the resulting liquid was added 50 ml dichloromethane andextracted with 3×50 ml 0.05 M HCl. The combined aqueous layer was madebasic with NaOH until the pH 10-11 and the solution was extracted with2×150 ml dichloromethane. The organic layers were combined and driedover anhydrous MgSO₄, filtered and evaporated to giveN-(4-methoxybenzyl)-1-(pyridin-4-yl)methanamine as a brown oil. Yield:0.36 g (38%) ¹H NMR (300 MHz, CDCl₃): δ 8.56 (2H, d), 7.28 (4H, m), 6.90(2H, m), 3.82 (5H, s), 3.76 (2H, s), ¹³C NMR (75 MHz, CDCl₃): δ 158.9,149.9, 149.6, 132, 129.4, 128.7, 123.1, 114, 65, 55.4, 52.7, 51.8.

Synthesis of Intermediate 8 Preparation ofN-benzyl-2,2-diphenylethanamine

To a round bottom flask with a magnetic stirrer bar benzylamine (0.86 g,8 mmol) and diphenylacetaldehyde (1.96 g, 10 mmol) were added togetherwith 10 ml methanol, NaCNBH₃ (0.64 g, 10 mmol) and a few granules of 4 Åmolecular sieve. The solution was stirred for 2-12 hours at roomtemperature and the progress of the reaction was monitored by thin layerchromatography (TLC). After filtration the mixture was concentrated invacuo and purified by flash chromatography using ethyl acetate/CH₂Cl₂(1:9) giving N-benzyl-2,2-diphenylethanamine as a viscous yellow oil.Yield: 0.45 g (20%), ¹H NMR (300 MHz, CDCl₃): δ 7.19 (15H, m), 4.16 (1H,m), 3.74 (2H, s), 3.18 (2H, m), ¹³C NMR (75 MHz, CDCl₃): δ 142.5, 139.7,128.3, 128.1, 128, 127.8, 126.7, 126.3, 125.8, 53.5, 50.9.

Example 2 Preparation ofN,N-bis(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine

To a round bottom flask with a magnetic stirrer bar 4-picolylamine (0.43g, 4 mmol) and 4-fluorobenzaldehyde (1.12 g, 9 mmol) were added togetherwith 10 ml methanol, NaCNBH₃ (0.57 g, 9 mmol) and a few granules of 4 Åmolecular sieve. The solution was stirred for 4 hours at roomtemperature and the progress of the reaction was monitored by thin layerchromatography (TLC). After filtration the mixture was concentrated invacuo and the resulting liquid was diluted with 50 ml dichloromethaneand extracted with 3×50 ml 0.05 M HCl. The combined aqueous layer wasmade basic with NaOH until the pH was 10-11 and the solution wasextracted with 2×150 ml dichloromethane. The organic layers werecombined and dried over anhydrous MgSO₄, filtered and evaporated to giveN,N-bis(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine as a yellow oil.Yield: 0.49 g (38%) ¹H NMR (300 MHz, CDCl₃): δ 8.53 (2H, d), 7.17 (10H,m), 3.76 (4H, d), 2.34 (2H, s), ¹³C NMR (75 MHz, CDCl₃): δ 163.4, 160.1,149.4, 148.9, 135.1, 135, 129.4, 129.3, 128.3, 122.7, 115.1, 114.8,64.1, 52.1, 51.4.

Example 3 Preparation of2,2′-(((pyridin-4-ylmethyl)azanediyl)bis(methylene))bis(benzene-1,4-diol)

To a round bottom flask with a magnetic stirrer bar 4-picolylamine (0.43g, 4 mmol) and 2,5-dihydroxybenzaldehyde (1.24 g, 9 mmol) were addedtogether with 10 ml methanol, NaCNBH₃ (0.57 g, 9 mmol) and a fewgranules of 4 Å molecular sieve. The solution was stirred for 4 hours atroom temperature and the progress of the reaction was monitored by thinlayer chromatography (TLC). After filtration the mixture wasconcentrated in vacuo and the resulting liquid was diluted with 50 mldichloromethane and extracted with 3×50 ml 0.05 M HCl. The combinedaqueous layer was made basic with NaOH until the pH was 10-11 and thesolution was extracted with 2×150 ml dichloromethane. The organic layerswere combined and dried over anhydrous MgSO₄, filtered and evaporated togive 2,2′-(((pyridin-4-ylmethyl)azanediyl)bis(methylene))bis(benzene-1,4-diol) as highly viscous dark brown oil.Yield: 0.88 g (62%), ¹H NMR (300 MHz, DMSO): δ 8.5 (5H, m), 7.35 (4H,m), 6.55 (8H, m), 3.69 (3H, d), 3.16 (4H, s), ¹³C NMR (75 MHz, DMSO): δ150.6, 150.4, 150, 149.3, 126.1, 124.8, 124.7, 123.9, 116.6, 116.5,116.3, 114.8, 60, 51.5, 49.8, 49.5.

Example 4 Preparation ofN,N-bis(4-fluorobenzyl)-1-(thiazol-2yl)methanamine

To a round bottom flask with a magnetic stirrer bar2-(aminomethyl)thiazole (0.46 g, 4 mmol) and 4-fluorobenzaldehyde (1.12g, 9 mmol) were added together with 10 ml methanol, NaCNBH₃ (0.57 g, 9mmol) and a few granules of 4 Å molecular sieve. The solution wasstirred overnight at room temperature and the progress of the reactionwas monitored by thin layer chromatography (TLC). After filtration themixture was concentrated in vacuo and the resulting liquid was dilutedwith 50 ml dichloromethane and extracted with 3×50 ml 0.05 M HCl. Thecombined aqueous layer was made basic with NaOH until the pH was 10-11and the solution was extracted with 2×150 ml dichloromethane. Theorganic layers were combined and dried over anhydrous MgSO₄, filteredand evaporated to giveN,N-bis(4-fluorobenzyl)-1-(thiazol-2-yl)methanamine as a dark brown oil,Yield: 0.91 g (69%), ¹H NMR (300 MHz, CDCl₃): δ 7.73 (1H, d), 7.32 (5H,m), 7.04 (4H, m), 4.67 (2H, s), 4.14 (2H, s), 3.86 (2H, s), ¹³C NMR (75MHz, CDCl₃): δ 171.7, 164. 163.8, 160.8, 160.6, 142.7, 135.5, 135,4,129.9, 129.8, 128.8, 119, 115,6, 115.5, 115.3, 115.2, 64.6, 52.5, 50.2.

Example 5 Preparation ofN,N-bis(4-methoxybenzyl)-1-(thiazol-2-yl)methanamine

To a round bottom flask with a magnetic stirrer bar2-(aminomethyl)thiazole (0.46 g, 4 mmol) and 4-methoxybenzaldehyde (1.22g, 9 mmol) were added together with 10 ml methanol, NaCN3H₃ (0.57 g, 9mmol) and a few granules of 4 Å molecular sieve. The solution wasstirred overnight at room temperature and the progress of the reactionwas monitored by thin layer chromatography (TLC). After filtration themixture was concentrated in vacuo and the resulting liquid was dilutedwith 50 ml dichloromethane and extracted with 3×50 ml 0.05 M HCl. Thecombined aqueous layer was made basic with NaOH until the pH was 10-11and the solution was extracted with 2×150 ml dichloromethane. Theorganic layers were combined and dried over anhydrous MgSO₄, filteredand evaporated to giveN,N-bis(4-methoxybenzyl)-1-(thiazol-2-yl)methanamine as a dark brownoil. Yield: 1 g (70%), ¹H NMR (300 MHz, CDCl,i): δ 7.73 (1H, d), 7.29(5H, m), 6.9 (4H, m), 4.62 (2H, s), 4.13 (2H, s), 3.81 (8H, s), ¹³C NMR(75 MHz, CDCl₃): δ 172, 159.2, 158.9, 142.6, 133.4, 131.8, 129.5, 128.7,118.9, 114, 65. 55.4, 52.7, 50.1.

Example 6 Preparation ofN-benzyl-N-4-methoxybenzyl)-1-(4-methoxyphenyl)methanamine

To a round bottom flask with a magnetic stirrer bar benzylamine (0.43 g,4 mmol) and 4-methoxybenzaldehyde (1.22 g, 9 mmol) were added togetherwith 10 ml methanol, NaCNBH₃ (0.57 g, 9 mmol) and a few granules of 4 Åmolecular sieve. The solution was stirred for 6 hours at roomtemperature and the progress of the reaction was monitored by thin layerchromatography (TLC). After filtration the mixture was concentrated invacuo and the resulting liquid was diluted with 50 ml dichloromethaneand extracted with 3×50 ml 0.05 M HCl. The combined aqueous layer wasmade basic with NaOH until the pH was 10-11 and the solution wasextracted with 2×150 ml dichloromethane. The organic layers werecombined and dried over anhydrous MgSO₄, filtered and evaporated to giveN-benzyl-N-(4-methoxybenzyl)-1-(4-methoxyphenyl)methanamine as a paleyellow oil. Yield: 0.29 g (20%) NMR (300 MHz, CDCl₃): δ 7.35 (9H, m),6.91 (4H, m), 3.8 (12H, m), ¹³C NMR (75 MHz, CDCl₃): δ 159.2, 140.9,133, 129.9, 129.2, 128.9, 128.7, 127.5, 114.5, 114.3, 65.6, 55.8, 53.6,53.1.

Example 7 Preparation of dimethyl4,4′-((benzylazanediyl)bis(methylene))dibenzoate

To a round bottom flask with a magnetic stirrer bar benzylamine (0.107g, 1 mmol) and methyl 3-formylbenzoate (0.49 g, 3 mmol) were addedtogether with 10 ml methanol, NaCNBH₃ (0.19 g, 3 mmol) and a fewgranules of 4 Å molecular sieve. The solution was stirred for 6 hours atroom temperature and the progress of the reaction was monitored by thinlayer chromatography (TLC). After filtration the mixture wasconcentrated in vacuo and the resulting liquid was diluted with 20 mldichloromethane and extracted with 3×20 ml 0.05 M HCl. The combinedaqueous layer was made basic with NaOH until the pH was 10-11 and thesolution was extracted with 3×60 ml dichloromethane. The organic layerswere combined and dried over anhydrous MgSO₄, filtered and evaporated togive dimethyl 4,4′-((benzylazanediyl)bis (methylene))dibenzoate as apale yellow oil. Yield: 0.14 g (18%), ¹³C NMR (75 MHz, CDCl₃): δ 169.3,167.1, 141.5, 140.7,140, 132.9, 131.5, 130.5, 130.4, 129.4, 128.9,128.7, 128.6, 128.4, 128.3, 128, 127.2, 64.8, 53.3, 52.8, 52.3, 52.4.

Example 8 Preparation of4-((benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline

To a round bottom flask with a magnetic stirrer bar benzylamine (0.43 g,4 mmol) and 4-(dimethylamino)benzaldehyde (1.6 g. 10.7 mmol) were addedtogether with 10 ml methanol, NaCNBH₃ (0.57 g, 9 mmol) and a fewgranules of 4 Å molecular sieve. The solution was stirred overnight atroom temperature and the progress of the reaction was monitored by thinlayer chromatography (TLC). After filtration the mixture wasconcentrated in vacuo and 100 mg of the crude product was purified byflash chromatography using ethyl acetate/CH₂Cl₂ (1:9) giving4-((benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline asa highly viscous brown oil. ¹³C NMR (75 MHz, CDCl₃): δ 150.3, 140.9,130.2, 129.3, 128.6, 128.3, 127.1, 113.1, 57,9, 57.5, 41.4.

Example 9 Preparation of4-(4-(dimethylamino)benzyl)(1-phenylethyl)amino)methyl)-N,N-dimethylaniline

To a round bottom flask with a magnetic stirrer bar 1-phenylethylamine(0.48 g, 4 mmol) and 4-(dimethylamino)benzaldehyde (1.34 g, 9 mmol) wereweighed in and added 10 ml methanol, NaCNBH₃ (0.57 g, 9 mmol) and a fewgranules of molecular sieve 4 Å. The solution was stirred for 6 hours atroom temperature and the progress of the reaction was monitored by thinlayer chromatography (TLC). After filtration the mixture wasconcentrated in vacuo and 100 mg of the crude product was purified byflash chromatography using ethyl acetate/CH₂Cl₂ (1:9) giving4-(((4-(dimethylamino)benzyl)(1-phenylethyl)amino)methyl)-N,N-dimethylanilineas a highly viscous brown oil. ¹H NMR (300 MHz, CDCl₃): δ 7.28 (10H, m),3.72 (2H, d), 3.84 (1H, q), 2.94 (6H, s), 2.19 (12H, s), 1.38 (3H, d),¹³C NMR (75 MHz, CDCl₃): δ 150, 146.1, 145.5, 129.3, 129.2, 128.6,128.4, 127.1, 126.9, 125.4, 112.9, 57.4, 51.1, 40.9, 25.5, 24.4.

Example 10 Preparation ofN-(4-(tert-butyl)benzyl)-N-(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine

To a round bottom flask with a magnetic stirrer bar Intermediate 1 (0.66g, 3 mmol) and 4-tert-butylbenzaldehyde (1.29 g, 8 mmol) were addedtogether with 10 ml methanol, NaCNBH₃ (0.50 g, 8 mmol) and a fewgranules of 4 Å molecular sieve. The solution was stirred for 8 hours atroom temperature and the progress of the reaction was monitored by thinlayer chromatography (TLC). After filtration the mixture wasconcentrated in vacuo and the resulting liquid was diluted with 30 mldichloromethane and extracted with 3×30 ml 0.05 M HCl. The combinedaqueous layer was made basic with NaOH until the pH was 10-11 and thesolution was extracted with 2×100 ml dichloromethane. The organic layerswere combined and dried over anhydrous MgSO₄, filtered and evaporated togiveN-(4-(tert-butyl)benzyl)-N-(4-fluorobenzyl)-1-(pyridin-4-yl)methanamineas a yellow oil. Yield: 0.19 g (18%), ¹H NMR (300 MHz, CDCl₃): δ 8.57(2H, d), 7.2 (10H, m), 3.81 (4H, d), 3.55 (2H, m), 1.3 (9H, m), ¹³C NMR(75 MHz, CDCl₃): δ 149.6, 149, 135.2, 129.4, 129.3, 126.6, 125.2, 125,123.3, 122.7, 115.1, 114.9, 52.1, 51.5, 31.1, 29.4.

Example 11 Preparation of2-(((4-fluorobenzyl)(pyridin-4-ylmethyl)amino)methyl)benzene-1,4-diol

To a round bottom flask with a magnetic stirrer bar Intermediate 3 (0.23g, 1 mmol) and 4-fluorobenzaldehyde (0.37 g, 3 mmol) were added togetherwith10 ml methanol, NaCNBH₃ (0.19 g, 3 mmol) and a few granules of 4 Åmolecular sieve. The solution was stirred for 8 hours at roomtemperature and the progress of the reaction was monitored by thin layerchromatography (TLC). After filtration the mixture was concentrated invacuo and the resulting liquid was diluted with 30 ml dichloromethaneand extracted with 3×30 ml 0.05 M HCl. The combined aqueous layer wasmade basic with NaOH until the pH was 10-11 and the solution wasextracted with 2×100 ml dichloromethane. The organic layers werecombined and dried over anhydrous MgSO₄, filtered and evaporated to give2-(((4-fluorobenzyl)(pyridin-4-ylmethyl)amino)methyl)benzene-1,4-diol asa brown oil. Yield: 0.39 g (100%), ¹ H NMR (300 MHz, DMSO): δ 8.51 (2H,d), 7.36 (4H, m), 7.14 (2H, m), 6.80 (1H, s), 6.60 (1H, s), 6.48 (1H,s), 3.32 (6H, s), ¹³C NMR (75 MHz, DMSO): δ 149.9, 149.6, 148.2, 148,138.7, 134.6, 130.5, 130.4, 128.4, 128.3, 124.4, 123.5, 115.7, 115.6,115.2, 114.9, 114.6, 114.3, 62.1, 56.5, 56, 52.1.

Example 12 Preparation of4-((benzyl(2,2-diphenylethyl)amino)methyl)-N,N-dimethylaniline

To a round bottom flask with a magnetic stirrer bar Intermediate 8 (0.24g, 0.83 mmol) and 4-(dimethylamino)benzaldehyde (0.18 g, 1.2 mmol) wereadded together with 10 ml methanol, NaCNBH₃ (0.10 g, 1.59 mmol) and afew granules of 4 Å molecular sieve. The solution was stirred for 6hours at room temperature and the progress of the reaction was monitoredby thin layer chromatography (TLC). After filtration the mixture wasconcentrated in vacuo and the crude product was purified by flashchromatography using ethyl acetate/CH₂Cl₂ (1:9) giving4-((benzyl(2,2-diphenylethyl)amino)methyl)-N,N-dimethylaniline as ayellow oil. ¹H NMR (300 MHz, CDCl₃): δ 7.15 (15H, m), 6.91 (2H, d), 6.57(2H, d), 4.17 H, t), 3.47 (4H, d), 2.96 (2H, d). 2.84 (6H, s).

Example 13 Preparation of4-((benzyl((2,3-dihydrobenzofuran-6-yl)methyl)amino)methyl)-N,Ndimethylaniline

To a round bottom flask with a magnetic stirrer bar Intermediate 6 (0.4g, 1.4 mmol) and 2,3-dihydrobenzofuran-5-carboxaldehyde (0.37 g, 2.5mmol) were added together with 10 ml methanol, NaCNBH₃ (0.16 g, 2.5mmol) and a few granules of 4 Å molecular sieve. The solution wasstirred for 8 hours at room temperature and the progress of the reactionwas monitored by thin layer chromatography (TLC). After filtration themixture was concentrated in vacuo and 100 mg of the crude product waspurified by flash chromatography using 5-10% ethyl acetate indichloromethane giving4-((benzyl((2,3-dihydrobenzofuran-6-yl)methyl)amino)methyl)-N,N-dimethylanilineas a viscous yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 7.22 (9H, m), 7.02(1H, d), 6.64 (2H, d), 4.47 (2H, t), 3.45 (2H, s), 3.38 (4H, s), 3.12(2H, t), 2.85 (6H, s), ¹³C NMR (75 MHz, CDCl₃): δ 159.4, 150.1, 140.5,132.2, 130, 129.1, 128.9, 128.5, 127.9, 127.1, 127, 125.8, 113, 109,71.6, 57.9, 57.7, 57.4, 51.2, 30.2.

Example 14 Preparation of methyl4-((benzyl(4-fluorobenzyl)amino)methyl)benzoate

First step:

To a round bottom flask with a magnetic stirrer bar benzylamine (2.14 g,10 mmol) and 4-fluorobenzaldehyde (1.55 g, 12.5 alma) were addedtogether with 10 ml methanol, NaCNBH₃ (0.79 g, 12.5 mmol) and a fewgranules of 4 Å molecular sieve. The solution was stirred for 4 hours atroom temperature and the progress of the reaction was monitored by thinlayer chromatography (TLC). After filtration the solution wasconcentrated in vacuo and the resulting liquid was diluted with 50 mldichloromethane and extracted with 3×50 ml 0.05 M HCl. The combinedaqueous layer was made basic with NaOH until the pH was 10-11 and thesolution was extracted with 2×150 ml dichloromethane. The organic layerswere combined and dried over anhydrous MgSO₄, filtered and evaporated togive N-benzyl-1-(4-fluorophenyl)methanamine.

Second step:

N-benzyl-1-(4-fluorophenyl)methanamine (0.61 g, 2,7 mmol) was added to20 ml acetone in a round bottom flask. To the solution methyl4-(bromomethyl)benzoate (1.15 g, 5 mmol) and K₂CO₃ (1.38 g, 10 mmol)were added and the reaction mixture was stirred for 4 hours at 55° C. atreflux. The progress of the reaction was monitored by thin layerchromatography (TLC), and when the reaction was complete the mixture wasfiltered and concentrated in vacuo. The crude product was purified byflash chromatography using 5-10% ethyl acetate in dichloromethane givingmethyl 4-((benzyl(4-fluorobenzyl)amino)methyl)benzoate as a pale yellowoil. ¹H NMR (300 MHz, CDCl₃): δ 7.92 (2H, d), 7.38 (2H, d), 7.24 (7H,m), 6.92 (2H, t), 3.83 (3H, s), 3.5 (2H, s), 3.46 (2H, s), 3.43 (2H, s).

Example 15 Preparation of 4-((benzyl(4-fluorobenzyl)amino)methyl)benzoicacid

The product from Example 13 was diluted in 0.1 M NaOH (15 ml) and addedto 10 ml methanol. The reaction mixture was stirred for 8 hours at 60°C. at reflux, and methanol was evaporated off on rotary evaporator. Theremaining aqueous phase was acidified with HCl until pH 5 and extractedwith 3×20 ml dichloromethane. The combined organic phase was dried overanhydrous MgSO₄ and concentrated in vacuo to give4-((benzyl(4-fluorobenzyl)amino)methyl)benzoic acid as a pale yellowoil. ¹H NMR (300 MHz, CDCl₃): δ 7.98 (2H, d), 7.41 (2H, d), 7.24 (7H,m), 6.92 (2H, t), 3.52 (2H, s), 3.46 (2H, s), 3.44 (2H, s).

Example 16 Preparation of1-(2′-(2H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl)-N-benzyl-N-(4-fluorobenzyl)methanamine

First step:

To a round bottom flask with a magnetic stirrer barN-benzyl-1-(4-fluorophenyl)methanamine (0.4 g, 1.97 mmol) and5-(4′-bromo-[1,1′-biphenyl]-2-yl)-2-trityl-2H-tetrazole (1.4 g, 2.5mmol) were added together with 10 ml acetone and K₂CO₃ (0.69 g, 5 mmol).The reaction mixture was stirred for 4 hours at 55° C. at reflux. Theprogress of the reaction was monitored by thin layer chromatography(TLC). After filtration the solution was concentrated in vacuo and thecrude product was purified by flash chromatography using 5-10% ethylacetate in dichloromethane giving 0.2 g of the tertiary amine.

Second step:

The tertiary amine from the first step of the reaction (0.2 g, 0.29mmol) was added 10 ml of a solution of trifluoro aceticacid/dichloromethane/methanol (2:1:1) and stirred for 3 hours at roomtemperature. The crude product was purified by flash chromatographyusing 5-10% ethyl acetate in dichloromethane giving1-(2′-(2H-tetrazol-5-yl)-[1,1′-blphenyl]-4-yl)-N-benzyl-N-(4-fluorobenzyl)methanamine as a pale brown oil. ¹H NMR (300 MHz, CDCl₃): δ 10.77 (1H,s), 8.08 (2H, d), 7.42 (10H, m), 7.03 (2H, t). 3.57 (6H, m), ¹³C NMR (75MHz, CDCl₃): δ 164, 160.7, 146.3, 139.4, 135.2, 130.6, 130,5, 129.1,129, 128.7, 127.5, 115,6, 115.4, 58.4, 58, 57.7.

Example 17 Synthesis of dimethyl4,4′-((benzylazanediyl)bis(methylene))dibenzoate

To a suspension of benzylamine (1.07 g, 10 mmol) and potassium carbonate(2.76 g, 20 mmol) in acetone (20 ml) was added methyl4-(bromomethyl)benzoate (4.58 g, 20 mmol) and the reaction mixture washeated to reflux overnight, cooled to room temperature and evaporated invacuo. The residue was separated with flash chromatography(dichloromethane) to give dimethyl4,4′-((benzylazanediyl)bis(methylene))dibenzoate as a brown viscous oil.Yield: 1.4 g (35%); TLC: R_(f): 0.85 (10% methanol in dichloromethane):¹H NMR (400 MHz, CDCl₃): δ 7.91 (4H, d), 7.38 (4H, d), 7.23 (5H, m),3.81 (6H, s), 3.51 (4H, s), 3.46 (2H, s); ¹³C NMR (400 MHz, CDCl₃): δ167.0, 144.9, 138.8, 129.7, 129.0, 128.8, 128.6, 128.3, 127.2, 58.2,57.8, 52.0 (¹³C NMR had some overlapping signals).

Example 18 Synthesis of 4,4′-((benzylazanediyl)bis(methylene)dibenzoicacid

Compound 17 (0.4 g, 1 mmol) was added to a suspension of potassiumhydroxide (0.17 g, 3 mmol) in 2 ml of 50:50 water/methanol. After 15minutes 1M HCl was added until the mixture became acidic and then washedwith water. The residue was subsequently suspended in toluene and driedin vacuo. Removal of toluene (as an azeotropic mixture with water)yielded 4,4′-((benzylazanediyl)bis(methylene)dibenzoic acid as lightbrown crystals. Yield: 0.21 g (56%); TLC: R_(f): 0.17 (10% methanol indichloromethane): ¹H NMR (400 MHz, DMSO): δ 12.83 (2H, s), 7.93 (4H, d),7.52 (4H, d), 7.38 (5H, m), 3.58 (4H, s), 3.52 (2H, s); ¹³C NMR (400MHz, DMSO): δ 167.1, 144.2, 138.6, 129.6, 129.4, 128.5, 128.3, 127.0,57.2, 56.8 (¹³C NMR had some overlapping signals).

Example 19 Synthesis ofN-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanamine

Benzylamine (0.536 g, 5 mmol), sodium hydrogen carbonate (0.92 g, 11mmol) and sodium dodecyl sulfate (approx. 20 mg) were added to water (20ml) in a 50 ml round-bottom flask, along with a stirring magnet. Themixture was heated at 80° C. for 5 min. 4-tert-Butylbenzyl bromide (2.5g, 11 mmol) was added to the mixture and subsequently heated to 80° C.for 1 hour. The reaction mixture was cooled to room temperature arid theresidue filtered and dried, before being recrystallized from a 1:1mixture of hexane and ethyl acetate to yieldN-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanamine asa white crystalline powder. Yield: 1.55 g (78%); TLC: R_(f): 0.88 (10%methanol in dichloromethane);¹H NMR (400 MHz, CDCl₃): δ 7.38 (13H, m),3.60 (6H, d), 1.34 (18H, s); ¹³C NMR (400 MHz, CDCl₃): δ 127.7, 127.3,126.6, 125.9, 124.7, 124.5, 124.3, 124.1 (¹³C NMR had some overlappingsignals).

Example 20 Synthesis ofN-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanamine

Benzylamine (0.536 g, 5 mmol), sodium hydrogen carbonate (0.92 g, 11mmol) and sodium dodecyl sulfate (approx. 20 mg) were added to water (20ml) in a 50 ml round-bottom flask, along with a stirring magnet. Themixture was heated at 80° C. for 5 min. 4-nitrobenzylbromide (2.37 g, 11mmol) was added to the mixture and subsequently heated to 80° C. for 1hour. The reaction mixture was cooled to room temperature and theresidue filtered and dried, before being recrystallized from a 1:1mixture of hexane and ethyl acetate to yieldN-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanamine as a whitecrystalline powder. Yield: 0.94 g (50%); TLC: R_(f)=0.86 (10% methanolin dichloromethane); ¹H NMR (400 MHz, CDCl₃): δ 8.21 (4H, d), 7.58 (4H,d), 7.32 (5H, m), 3.69 (4H, s), 3.60 (2H, s); ¹³C NMR (400 MHZ, CDCl₃):δ 147.3, 146.8, 138.0, 129.2, 128.7, 128.6, 127.6, 123.7, 58.5, 57.6.

Example 21 Synthesis of tris(4-nitrobenzyl)amine

A mixture of 4-nitrobenzyl bromide (6.48 g, 30 mmol) and aqueous ammoniasolution (30-33%, 2 ml) in methanol (15 ml) was heated in a sealed tubeat 100° C. for 4 hours. Then it was cooled to room temperature andpoured into water (80 ml). The resulting mixture was extracted threetimes with dichloromethane and the combined organic layers were driedover sodium sulfate. Removal of the solvents yielded a yellow solid,which was recrystallized in ethyl acetate to givetris(4-nitrobenzyl)amine as a light yellow crystal. Yield: 0.63 g (15%);TLC: R_(f): 0.87 (10% methanol in dichloromethane); ¹H NMR (400 MHz,CDCl₃): δ 8.25 (6H, d), 7.56 (6H, d), 3.70 (6H, s); ¹³C NMR (400 MHz,CDCl₃): δ 147.5, 145.8, 129.2, 123.9, 57.8.

Example 22 Synthesis of 4-(((4-aminobenzyl)(benzyl)amino)methyl)aniline

N-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanamine (compound 20)(2.19 g, 5.81 mmol), calcium chloride (0.782 g, 5.32 mmol) andacid-washed zinc (15 g) were suspended in 125 ml of ethanol. The mixturewas refluxed for 2 hours, cooled to room temperature, filtered and driedin vacuo, before being recrystallized from a 1:1 mixture of hexane anddiethyl ether to yield 4-(((4-aminobenzyl)(benzyl)amino)methyl)anilineas a light yellow crystalline powder. Yield: 0.37 g (20%); TLC: R_(f):0.51 (10% methanol in dichloromethane); ¹H NMR (400 MHz, DMSO): δ 7.33(5H, m), 7.21 (4H, d), 6.97 (4H, d), 4.90 (4H, s), 3.40 (2H, s), 3.27(4H, s); ¹³C NMR (400 MHz, DMSO): δ 147.3, 139.9, 129.2, 128.3, 128.1,126.7, 125.4, 113.7, 56.4, 48.6.

Example 23 Synthesis of4-((benzyl(4-(diethylamino)benzy)amino)methyl)-N,N-diethylaniline

A suspension of4-((benzyl(4-(diethylamino)benzyl)amino)methyl)-N,N-diethylaniline(compound 22) (0.6 g, 1.9 mmol) and potassium carbonate (2.76 g, 20mmol) in acetone (50 ml) was added ethyl iodide (3.92 g, 20 mmol) andthe reaction mixture was heated to reflux overnight, cooled to roomtemperature and evaporated in vacuo The residue was separated with flashchromatography (10% methanol in dichloromethane) to give4-((benzyl(4-(diethylamino)benzyl)amino)methyl)-N,N-diethylaniline as aviscous brown oil. Yield: 0.05 g (6.1%-NB ¹H NMR indicated that thesubstance was not completely dry); TLC: R_(f): 0.82 (10% methanol indichloromethane); ¹H NMR (300 MHz, CDCl₃): δ 7.40 (9H, m), 6.74 (4H, m),3.66 (2H, s), 3.58 (4H, s), 3.42 (8H, q), 1.25 (12H, t).

Example 24 Synthesis of 4-((bis(4-aminobenzyl)amino)methyl)aniline

A mixture of tris(4-nitrobenzyl)amine (compound 21) (848 mg, 2 mmol),10% Pd-C (42.4 mg) and aqueous HCl (37%, 4 ml) in methanol (30 ml) washydrogenated at room temperature and atmospheric hydrogen pressure for 3hours. After the catalyst was filtered off and methanol was evaporated,the residue was dissolved in ethanol and toluene. Removal of thesesolvents (as azeotropic mixtures with water) yielded a light yellowsolid which was washed with diethyl ether to give4-((bis(4-ammoniobenzyl)ammonio)methyl)benzenaminium as light yellowcrystals. Yield: 0.56 g (84% NB the NMR indicated that the substance wasnot completely dried); TLC: R_(f): 0.16 (10 methanol indichloromethane); ¹H NMR (300 MHz, D₂O): δ 7.39 (6H, d), 7.33 (6H, d),4.33 (6H, s).

Example 25 Synthesis of1,1′-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(p-tolyl)urea)

4-(((4-Aminobenzyl)(benzyl)amino)methyl)aniline (compound 22) (0.1 g,0.315 mmol) was dissolved in dry dichloromethane (4 ml) and p-tolylisocyanate (0.084 g, 0.63 mmol) was slowly added at 0° C. After stirringat room temperature for 18 hours, the solvent was removed and diethylether (3 ml) was added. The white solid was filtered off and dried toyield1,1′-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(p-tolypurea)as a white powder. Yield: 0.147 g (80%); TLC: R_(f): 0.54 (10% methanolin dichloromethane); ¹H NMR (400 MHz, DMSO): δ 8.54 (4H, d), 7.36 (17H,m), 7.07 (4H, d), 3.45 (6H, d), 2.24 (6H, s); ¹³C NMR (400 MHz, DMSO): δ152,5, 138.6, 137.1, 132.2, 130.5, 129.1, 128.9, 128.4, 128.2, 118.2,118.1, 56.7, 56.3, 20.3 (¹³C NMR had some overlapping signals).

Example 26 Synthesis of1,1′-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(4-fluorophenyl)urea)

4-(((4-Aminobenzyl)(benzyl)amino)methyl)aniline (compound 22) (0.1 g,0.315 mmol) was dissolved in dry dichloromethane (4 ml) and4-fluorophenyl isocyanate (0.084 g, 0.63 mmol) was slowly added at 0° C.After stirring at room temperature for 18 hours, the solvent was removedand diethyl ether (3 ml) was added. The white solid was filtered off anddried to yield1,1′-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(4-fluorophenyl)urea)as a white powder. Yield 0.14 g (75%); TLC: R_(f): 0.59 (10% methanol indichloromethane); ¹H NMR (400 MHz, DMSO): δ 8.66 (4H, d), 7.27 (21H, m),3.48 (2H, s), 3.42 (4H, s); ¹³C NMR (300 MHz, DMSO): δ 153.5, 139.3,136.94, 136.90, 133.2, 129.8, 129.3, 129.1, 120.8, 120.6, 119.1, 116.3,116.0, 80.3, 80.1 (³C NMR had some overlapping signals).

Example 27 Synthesis of1,1′-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-phenylurea)

4-(((4-Aminobenzyl)(benzyl)amino)methyl)aniline (compound 22) (0.1 g,0.315 mmol) was dissolved in dry dichloromethane (4 ml) and phenylisocyanate (0.075 g, 0.63 mmol) was slowly added at 0° C. After stirringat room temperature for 18 hours, the solvent was removed and diethylether (3 ml) was added. The white solid was filtered off and dried toyield1,1′-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-phenylurea)as a white powder. Yield: 0.08 g (46%); TLC; R_(f): 0.76 (10% methanolin dichloromethane); ¹H NMR (400 MHz, DMSO): δ 8,61 (4H, s), 7.22 (23H,m), 3.49 (4H, s). 3.43 (2H, s); ¹³C NMR (400 MHz, DMSO)): δ 152.5,139.7, 139.3, 138.5, 132.4, 128.9, 128.7, 128.4, 128.2, 126.8, 121.7,118.1, 56.7, 56.3.

Example 28 Synthesis of1,1′,1″-((nitrilotris(methylene))tris(benzene-4,1-diyl))tris(3-(p-tolypurea)

4-((Bis(4-ammoniobenzyl)ammonio)methyl)benzenaminium (compound 24) (100mg, 0.297 mmol) was dissolved in water and stirred at room temperature,before potassium carbonate was added until the pH became 11-12. Themixture was extracted three times with dichloromethane and subsequentlydried in vacuo. The residue was dissolved in dry dichloromethane (4 ml)and p-tolyl isocyanate (0.136 g, 0.9 mmol) was slowly added at 0° C.After stirring at room temperature for 18 hours, the solvent was removedand diethylether (3 ml) was added. The white solid was filtered off togive1,1′,1″-((nitrilotris(methylene))tris(benzene-4,1-diyl))tris(3-(p-tolyl)urea)as a white powder. Yield: 0.07 g (32%); TLC: R_(f): 0.76 (10% methanolin dichloromethane); ¹H NMR (300 MHz, DMSO): δ 8.57 (6H, d), 7.25 (24H,m), 3.41 (6H, s), 2.24 (9H, s).

Example 29 Synthesis of4-((benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline

A solution of benzylamine (1.29 g, 12 mmol) and4-(dimethylamino)benzaldehyde (3.75 g, 25 mmol) in methanol (50 ml) wasstirred for 30 minutes before sodium cyanoborohydride (1.6 g, 25 mmol)and 4 Å molecular sieves were added. The mixture was stirred overnight,filtered and thereafter dried in vacuo. The residue was separated withflash chromatography (5-10% ethyl acetate in dichloromethane) to give4-((benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline asa brown viscous oil. Yield: 1.6 g (35%); TLC: R_(f): 0.83 (10% methanolin dichloromethane); ¹H NMR (400 MHz, CDCl₃): δ 7.32 (2H, m), 7.18 (7H,m), 6.63 (4H, d), 3.45 (2H, s), 3.38 (4H, s), 2.84 (12H, 5); ¹³C NMR(400 MHz, CDCl₃): δ 149.7, 140.3, 129.7, 128.7, 128.0, 127.7, 126.5,112.7, 57.4, 57.0, 40.8.

Example 30 Synthesis ofN-benzyl-N-(4-(methoxycarbonyl)benzyl)-1-(4(methoxycarbonyl)phenyl)methanaminiumchloride

Dimethyl 4,4′-((benzylazanediyl)bis(methylene))dibenzoate (compound 17)(0.2 g, 0.5 mmol) was added to stirring diethyl ether in a round bottomflask and 2.0 M hydrochloride solution in diethyl ether (0.3 mL, 0.6mmol) was slowly added. After 5 minutes, the precipitate was filteredoff and dried under vacuum to yieldN-benzyl-N-(4-(methoxycarbonyl)benzyl)-1-(4-(methoxycarbonyl)phenyl)methanaminiumchloride as a light brown powder.

Example 31 Synthesis ofN-benzyl-N-(4-(methoxycarbonyl)benzyl)-1-(4-(methoxycarbonyl)phenyl)methanaminiummethanesulfonate

Dimethyl 4,4′-((benzylazanediyl)bis(methylene))dibenzoate (compound 17)(0.2 g, 0.5 mmol) was added to stirring diethyl ether in a round bottomflask and methane sulfonic acid (0.05 g, 0.52 mmol) was slowly added.After 5 minutes, the precipitate was filtered off and dried under vacuumto yieldN-benzyl-N-(4-(methoxycarbonyl)benzyl)-1-(4-(methoxycarbonyl)phenyl)methanaminiummethanesulfonate as a brown viscous substance.

Example 32 Synthesis ofN-benzyl-N-(4-carboxybenzyl)-1-(4-carboxyphenyl)methanaminium chloride

4,4′-((Benzylazanediyl)bis(methylene)dibenzoic acid (compound 18) (0.2g, 0.53 mmol) was added to stirring diethyl ether in a round bottomflask and 2.0 M hydrochloride solution in diethyl ether (0.3 mL, 0.6mmol) was slowly added. After 5 minutes, the precipitate was filteredoff and dried under vacuum to yieldN-benzyl-N-(4-carboxybenzyl)-1-(4-carboxyphenyl)methanaminium chlorideas a light grey crystal-like powder.

Example 33 Synthesis ofN-benzyl-N-(4-carboxybenzyl)-1-(4-carboxyphenyl)methanaminiummethanesulfonate

4,4′-((Benzylazanediyl)bis(methylene)dibenzoic acid (compound 18) (0.2g, 0.53 mmol) was added to stirring diethyl ether in a round bottomflask and methane sulfonic acid (0.06 g, 0.62 mmol) was slowly added.After 5 minutes, the precipitate was filtered off and dried under vacuumto yield N-benzyl-N-(4-carboxybenzyl)-1-(4-carboxyphenyl)methanaminiummethanesulfonate chloride as a red viscous substance.

Example 34 Synthesis ofN-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanaminiumchloride

N-Benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanamine(compound 19) (0.2 g, 0.46 mmol) was added to stirring diethyl ether ina round bottom flask and 2.0 M hydrochloride solution in diethyl ether(0.25 mL, 0.5 mmol) was slowly added. After 5 minutes, the precipitatewas filtered off and dried under vacuum to yield ofN-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanaminiumchloride as a white crystal-like powder.

Example 35 Synthesis ofN-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanaminiummethanesulfonate

N-Benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanamine(compound 19) (0.2 g, 0.46 mmol) was added to stirring diethyl ether ina round bottom flask and methane sulfonic acid (0.05 g, 0.52 mmol) wasslowly added. After 5 minutes, the precipitate was filtered off anddried under vacuum to yieldN-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanaminiummethanesulfonate as a viscous substance.

Example 36 Synthesis ofN-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanaminium chloride)

N-Benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanamine (compound 20)(0.2 g, 0.53 mmol) was added to stirring diethyl ether in a round bottomflask and 2.0 M hydrochloride solution in diethyl ether (0.3 mL, 0.6mmol) was slowly added. After 5 minutes, the precipitate was filteredoff and dried under vacuum toN-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanaminium chloride as alight yellow crystal-like powder.

Example 37 Synthesis ofN-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanaminiummethanesulfonate

N-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanamine (compound 20)(0.2 g, 0.53 mmol) was added to stirring diethyl ether in a round bottomflask and methane sulfonic acid (0.06 g, 0.62 mmol) was slowly added.After 5 minutes, the precipitate was filtered off and dried under vacuumto yieldN-benzyl-N-(4-(dimethylamino)benzyl)-1-(4-(dimethylamino)phenyl)methanaminiummethanesulfonate as a light yellow viscous substance.

Example 38 Synthesis of N,N-bis(4-aminobenzyl)-1-phenylmethanaminiumchloride

4-(((4-Aminobenzyl)(benzyl)amino)methyl)aniline (compound 22) (0.2 g,0.63 mmol) was added to stirring diethyl ether in a round bottom flaskand 2.0 M hydrochloride solution in diethyl ether (0.35 mL, 0.7 mmol)was slowly added. After 5 minutes, the precipitate was filtered off anddried under vacuum to yield N,N-bis(4-aminobenzyl)-1-phenylmethanaminiumchloride as a light yellow crystal-like powder.

Example 39 Synthesis of N,N-bis(4-aminobenzyl)-1-phenylmethanaminiummethanesulfonate

4-(((4-Aminobenzyl)(benzyl)amino)methylaniline (compound 22) (0.2 g,0.63 mmol) was added to stirring diethyl ether in a round bottom flaskand methane sulfonic acid (0.07 g, 0.73 mmol) was slowly added. Alter 5minutes, the precipitate was filtered off and dried under vacuum toyield N,N-bis(4-aminobenzyl)-1-phenylmethanaminium methanesulfonate as alight yellow viscous substance.

Example 40 Synthesis ofN-benzyl-N-(4-(diethylamino)benzyl)-1-(4-(diethylamino)phenyl)methanaminiumchloride

4-((Benzyl(4-(diethylamino)benzyl)amino)methyl)-N,N-diethylaniline(compound 23) (0.2 g, 0.47 mmol) was added to stirring diethyl ether ina round bottom flask and 2.0 M hydrochloride solution in diethyl ether(0.25 mL, 0.5 mmol) was slowly added. After 5 minutes, the precipitatewas filtered off and dried under vacuum to yieldN-benzyl-N-(4-(diethylamino)benzyl)-1-(4-(diethylamino)phenyl)methanaminiumchloride as a light yellow crystal-like powder.

Example 41 Synthesis ofN-benzyl-N-(4-(diethylamino)benzyl)-1-(4-(diethylamino)phenyl)methanaminiummethanesulfonate

4-((Benzyl(4-(diethylamino)benzyl)amino)methyl)-N,N-diethylaniline(compound 23) (0.2 g, 0.47 mmol) was added to stirring diethyl ether ina round bottom flask and methane sulfonic acid (0.25 mL, 0.5 mmol) wasslowly added. After 5 minutes, the precipitate was filtered off anddried under vacuum to yieldN-benzyl-N-(4-(diethylamino)benzyl)-1-(4-(diethylamino)phenyl)methanaminiummethanesulfonate as a light yellow viscous substance.

Example 42 Synthesis ofN-benzyl-N-(4-(3-(p-tolyl)ureido)benzyl)-1-(4-(3-(p-tolypureido)phenyl)methanaminiumchloride

1,1′-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(p-tolyl)urea)(compound 25) (0.2 g, 0.34 mmol) was added to stirring diethyl ether ina round bottom flask and 2.0 M hydrochloride solution in diethyl ether(0.2 mL, 0.4 mmol) was slowly added. After 5 minutes, the precipitatewas filtered off and dried under vacuum to yieldN-benzyl-N-(4-(3-(p-tolyl)ureido)benzyl)-1-(4-(3-(p-tolypureido)phenyl)methanaminiumchloride as a yellow crystal-like powder.

Example 43 Synthesis ofN-benzyl-N-(4-(3-(p-tolypureido)benzyl)-1-(4-(3-(p-tolyl)ureido)phenyl)methanaminiummethanesulfonate

1,1′-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(p-tolyl)urea)(compound 25) (0.2 g, 0.34 mmol) was added to stirring diethyl ether ina round bottom flask and methane sulfonic acid (0.04 g, 0.42 mmol) wasslowly added. After 5 minutes, the precipitate was filtered off anddried under vacuum to yieldN-benzyl-N-(4-(3-(p-tolyl)ureido)benzyl)-1-(4-(3-(p-tolyl)ureido)phenyl)methanaminiummethanesulfonate as a yellow powder.

Example 44 Synthesis ofN-benzyl-N-(4-(3-(4-fluorophenyl)ureido)benzyl)-1-(4-(3-(4-fluorophenyl)ureido)phenyl)methanaminiumchloride

1,1′-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(4-fluorophenyl)urea)(compound 26) (0.2 g, 0.34 mmol) was added to stirring diethyl ether ina round bottom flask and 2.0 M hydrochloride solution in diethyl ether(0.2 mL, 0.4 mmol) was slowly added. After 5 minutes, the precipitatewas filtered off and dried under vacuum to yieldN-benzyl-N-(4-(3-(4-fluorophenyl)ureido)benzyl)-1-(4-(3-(4-fluorophenyl)ureido)phenyl)methanaminiumchloride as a yellow powder.

Example 45 Synthesis ofN-benzyl-N-(4-(3-(4-fluorophenyl)ureido)benzyl)-1-(4-(3-(4-fluorophenyl)ureido)phenyl)methanaminiummethanesulfonate

1,1′-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(4-fluorophenyl)urea)(compound 26) (0.2 g, 0.34 mmol) was added to stirring diethyl ether ina round bottom flask and methane sulfonic acid (0.04 g, 0.42 mmol) wasslowly added. After 5 minutes, the precipitate was filtered off anddried under vacuum to yieldN-benzyl-N-(4-(3-(4-fluorophenyl)ureido)benzyl)-1-(4-(3-(4-fluorophenyl)ureido)phenyl)methanaminiummethanesulfonate as a yellow substance.

Example 46 Synthesis ofN-benzyl-N-(4-(3-phenylureido)benzyl)-1-(4-(3-phenylureido)phenyl)methanaminiumchloride

1,1′-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-phenylurea)(compound 27) (0.2 g, 0.36 mmol) was added to stirring diethyl ether ina round bottom flask and 2.0 M hydrochloride solution in diethyl ether(0.2 mL, 0.4 mmol) was slowly added. After 5 minutes, the precipitatewas filtered off and dried under vacuum to yieldN-benzyl-N-(4-(3-phenylureido)benzyl)-1-(4-(3-phenylureido)phenyl)methanaminiumchloride as a yellow powder.

Example 47 Synthesis ofN-benzyl-N-(4-(3-phenylureido)benzyl)-1-(4-(3-phenylureido)phenyl)methanaminiummethanesulfonate

1,1′-4(Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-phenylurea)(compound 27) (0.2 g, 0.36 mmol) was added to stirring diethyl ether ina round bottom flask and methane sulfonic acid (0.04 g, 0.42 mmol) wasslowly added. After 5 minutes, the precipitate was filtered off anddried under vacuum to yieldN-benzyl-N-(4-(3-phenylureido)benzyl)-1-(4-(3-phenylureido)phenyl)methanaminiummethanesulfonate as a viscous yellow substance.

Example 48 Synthesis ofN-benzyl-N-(4-(dimethylamino)benzyl)-1-(4-(dimethylamino)phenyl)methanaminiumchloride

4-(Benzyl(4-(dimethylamino)benzy)amino)methyl)-N,N-dimethylaniline(compound 29) (0.2 g, 0.54 mmol) was added to stirring diethyl ether ina round bottom flask and 2.0 M hydrochloride solution in diethyl ether(0.3 mL, 0.6 mmol) was slowly added. After 5 minutes, the precipitatewas filtered off and dried under vacuum to yieldN-benzyl-N-(4-(dimethylamino)benzyl)-1-(4-(dimethylamino)phenyl)methanaminiumchloride as a light yellow crystal-like powder.

Example 49 Synthesis ofN-benzyl-N-(4-(dimethylamino)benzyl)-1-(4-(dimethylamino)phenyl)methanaminiummethanesulfonate

4-((Benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline(compound 29) (0.2 g, 0.54 mmol) was added to stirring diethyl ether ina round bottom flask and methane sulfonic acid (0.06 g, 0.62 mmol) wasslowly added. After 5 minutes, the precipitate was filtered off anddried under vacuum to yieldN-benzyl-N-(4-(dimethylamino)benzyl)-1-(4-(dimethylamino)phenyl)methanaminiummethanesulfonate as a light brown viscous substance.

Example 50 Aqueous Solubility of Free Bases and Various Salts

The aqueous solubility of various amine compounds and theircorresponding salts were determined by dissolution of various amounts ofthe respective compounds at room temperature.

Solubility Solubility free base Solubility Mesylate Compound (mg/ml) HClsalt salt 17 <0.36 <0.36 <0.36 18 <0.05 <0.05 <0.05 19 <0.05 <0.05 <0.0520 <3 <3 <3 22 <0.05 4.5 >22 23 <0.05 15 >34 25 <0.05 <0.05 <0.05 26<1.5 <1.5 <1.5 27 <0.5 <0.5 <0.5 29 <0.16 6 94

Example 51 Aqueous Solubility of Tribenzylamine as Free Bases andVarious Salts

Tribenzylamine and the corresponding salts were dissolved in water andthe solubility was quantified (area under curve (AUC)) by HPLC

Solubility of tribenzylamine and its corresponding HCl and mesylatesalts, tested on HPLC. Free Mesylate base HCl salt salt Compound (mAu*s)(mAu*s) (mAu*s) AUC ≈0 406 1975

Examples 52-54 Pharmacological Testing of Newly Synthesized CompoundsExample 52 Testing of Derivatized and Newly Synthesized Tertiary Aminesin the AKAP18δ-PLB AlphaScreen Assay

The compounds of formula (I) and (I′) were made in two groups thatdiffered in their substituents on the aromatic rings. Tertiary amineswhere two of three substituents were similar were defined in one group(Series 2, 2a-2h), while those containing three unique substituents werein the other (Series 3, 3a-3g).

Two compounds in the group 2a-2h were found to have particularly lowEC₅₀ values. Compound 2b contained four hydrogen-bond donors and twohydrogen-bond acceptors, increasing the possibility of binding to theprotein complex through hydrogen binding. In compound 2g two nitrogenswere introduced in substituents coupled to the aromatic rings, leavingthe molecule with three hydrogen-binding domains. FIGS. 2 and 3 show theconcentration-response curves of compounds 2b and 2g in the AKAP18δassay.

In the last group of derivatized compounds, the central nitrogen hadthree different substituents, and by using fragments of the compoundsgiving a low EC₅₀ value further highly active compounds where obtained.Compound 3b was based on the structure of 2b, having one substituentwith hydroxyl groups, and the other with fluorine in para-position. TheEC₅₀ value of 3b was approximately the same as for 2b, as shown in FIG.4.

Two compounds were synthesized based on 2g, keeping one of the twodimethylamino substituents. In 3c the other part of the molecule was adiphenyl group, a hydrophobic moiety like the one to be found in some ofthe original compounds. This resulted in an EC₅₀ value of 0.327 μM andwas the most potent compound described. Compound 3d also contained thedimethylamino group, this time in combination with a benzofuran. Thiscompound had an EC₅₀ of 2.79 μM. The concentration response curves ofcompounds 3c and 3d are shown in FIGS. 5 and 6.

In order to find out whether an acidic moiety was favorable, a compoundcontaining both a basic moiety and a carboxylic acid was synthesised.The synthesis started with making the methylether. After completion ofthe reaction, a small amount of compound 3e was isolated to beforehydrolysis to provide compound 3f. In 3f a carboxylic acid wasintroduced on the benzene ring, giving the molecule both basic-andacidic properties, like the original group of analogues had. However,compound 3e (EC₅₀ 34.84 μM) and compound 3f (36.55 μM) were found tohave slightly higher EC₅₀ values.

In the last compound synthesized another acidic group was introduced,this time a tetrazole. The tetrazole is a part of the pharmacophore of agroup of existing drugs, angiotensin-II blockers, commonly used in thetreatment of hypertension.

The rationale for introducing this group was that it had the acidicproperties similar to the originally identified compound, and earliertesting had shown that substituents containing nitrogen seemed to bepreferable in terms of low EC₅₀. In this case, four nitrogens wereintroduced to the molecule, making molecule able to act both as hydrogenbinding acceptor and donor. In addition to this an extra benzene ringwas added, giving the molecule a larger hydrophobic site. However,compound 3g was found to have the highest EC₅₀ value of this screen with47.07 μM.

Example 53 Cell-Based Testing of Derivatized Compounds in Cardiomyocytes

Based on data from studies in cardiomyocytes assay using a peptide thatblocks AKAP18δ-PLB interaction, it was expected that effective compoundswould also cause disruption of the AKAP18δ-PLB complex and decreasePLB-Ser¹⁶ phosphorylation (Lygren et al. in EMBO Reports, volume 8,issue 11, page 1061-1067 (2007). A small selection of hits from thederivatized compounds were tested in this cell-based assay, performed asdescribed in Lygren et al.

Briefly, compounds were added to primary cultures of rat neonatalcardiomyocytes were added compounds about 24 hour prior stimulation withisoproterenol, and amount of phosphorylated Ser¹⁶-PLB was analyzed bywestern blotting using a pSer¹⁶-PLB phosphospecific antibody. Actin wasused as a loading control.

Four synthesized compounds were tested, as shown below:

Compound 2g displayed a concentration dependent inhibition ofPLB-phosphorylation. Even though compound 3c had the lowest EC₅₀ valueof the synthesized compounds in vitro, the effect on PLB phosphorylationin cardiomyocytes was somewhat weaker than that of 2g. Compounds 3d and3f were tested in the same experiment, but did not appear to have aneffect on the phosphorylation of PLB in this assay.

The results are shown in FIG. 7. Rat neonatal cardiomyocytes treatedwith compounds 24 hours prior stimulation with isoproterenol (100 nM, 5minutes). The histogram shows levels of phosphorylated Ser¹⁶-PLBquantified by densiometery relative to actin levels.

Example 54 Cell Viability Assay on Derivatized Compounds

Compound toxicity was measured in a luminescence assay that gives asignal proportional to the amount of metabolically active cells in thesample; the assay was performed as described by the supplier. Allsynthesized compounds were tested in the viability assay, and viabilitywas registered at 24 hours and 72 hours. The results after 24 hoursincubation with the synthesised compounds is shown in FIG. 8.

Examples 52-54 Summary of Test Results on Derivatized Compounds

TABLE 2 Overview of effect and viability of synthesized tertiary amineswith two different substituents. EC₅₀ average is given with ±standarddeviation (n = 3) or ±half range (n = 2). − = no activity, + = possibleeffect, ++ = effect, n.d = not done Effect on PLB EC₅₀ phosphorylationViability in μM in (100 μM) Compound Structure (n) cardiomyocytes 24hours 2a

− n.d 0.85 2b

12.33 ± 3.08 (2) − 0.10 2c

− n.d 1.11 2d

− n.d 1.15 2e

− n.d 1.01 2f

− n.d 0.92 2g

1.7 ± 0.91 (3) ++ 0.56 2h

− n.d 0.91

TABLE 3 Overview of effect and viability of synthesized tertiary amineswith three different substituents. EC₅₀ average is given with ±standarddeviation (n = 3) or ±half range (n = 2). − = no activity, + = possibleeffect, ++ = effect, n.d = not done Effect on PLB phosphorylationViability EC₅₀ in (100 μM) Compound Structure (n) cardiomyocytes 24hours 3a

− n.d 0.79 3b

5.43 ± 1.11 (2) n.d 0.03 3c

0.44 ± 0.18 (3) + 0.74 3d

2.45 ± 0.73 (3) − 0.58 3e

34.84 (1) n.d 0.57 3f

40.56 ± 7.4 (3) + 0.98 3g

47.07 (1) n.d 0.80

Example 55

Following on Example 53, two compounds, 2g and 3d (FIG. 9A,B and 9C,respectively), were tested in more detail for their ability to interferewith phosphorylation of phospholamban after stimulation of neonatal(FIG. 9A and 9C) or adult (FIG. 9B) primary cultures of cardiomyocytes.As can be seen from the Figure, compound 2g inhibited phospholambanphosphorylation with a half-maximal effect of approximately 40 and 100μM in neonatal and adult cardiomyocytes, respectively whereas compound3d had a half-maximal effect of approximately 100 μM in neonatal cells.

Example 56

Adult rat cardiomyocytes were isolated and perfused with externalsolution (NaCl 125, CsCl 20, D-glucose 5, MgCl₂ 1, CaCl₂ 1.8, Hepes 10,4-aminopyridine 5, probenecid 2, pH 7.4 by NaCH). Cell dialysis wasachieved by 1-2 MΩ patch pipettes, filled with (in mM): CsCl 115, TEACl20, Hepes 10, MgATP 5, Na2-Phosphocreatinine 5, EGTA 0.04, cAMP 0.005,adjusted to pH=7.2 with CsOH. Compound 2g (mesylate salt dissolved inwater) was included at a concentration of 10 uM. Whole-cell Cafluorescence measured from voltage-clamped cells was performed withCairn Research Optoscan Monochromator (Excitation 488 nm, emission 515nm long pass) (Cain Research Ltd., Faverham, UK). By applying 100 mssquare voltage step from −45 to 0 mV at 0.125 Hz by an Axociamp 2Bamplifier (Axon Instruments, Foster City, Calif., USA), basal Catransients were recorded, and tau values were obtained bymonoexponential fitting of the Ca²⁺ extrusion phase from regulartransients (τ) and caffeine transients (τcaff) after rapid applicationsof 10 mM caffeine. SERCA2 rate constant were calculated as:k_SERCA=1/τ−1/τcaff.

As can been seen from the figure, incubation with compound 2g in thepresence of cAMP increased SERCA2 activity leading to a faster calciumreuptake in sarcoplasmic reticulum and relaxation of the heart to allowfilling. This indicates an ability of this class of compounds toregulation SERCA2 activity, positively or negatively, possibly dependingon concentration or other aspects of the structure activityrelationship.

1-27. (canceled)
 28. A compound of formula (I) or a pharmaceuticallyacceptable salt thereof:

wherein L¹, L² and L³ independently denote C₁-C₄-alkylene optionallysubstituted with one phenyl; Ar¹ denotes phenyl substituted with one ormore R; Ar² denotes phenyl substituted with one or two R; Ar³ denotesphenyl; R denotes NR²R³; and R² and R³ independently denote H orC₁-C₄-alkyl.
 29. The compound of claim 28, wherein L¹, L² and L³independently denote C₁-C₄-alkylene.
 30. The compound of claim 28,wherein L¹, L² and L³ denote —CH₂—.
 31. The compound of claim 28,wherein Ar¹ denotes phenyl substituted with one R.
 32. The compound ofclaim 28, wherein Ar² denotes phenyl substituted with one R.
 33. Thecompound of claim 28, wherein R² and R³ independently denoteC₁-C₄-alkyl.
 34. The compound of claim 28, wherein R² and R³ denotemethyl.
 35. The compound of claim 28, wherein L¹, L² and L³ denote—CH₂—, Ar¹ denotes phenyl substituted with one R, Ar² denotes phenylsubstituted with one R; and R² and R³ independently denote C₁-C₄-alkyl.36. The compound of claim 28 having the following structure:


37. The compound of claim 28 having the following structure:


38. The compound of claim 28 having the following structure: